Vectors and methods for gene transfer

ABSTRACT

The present invention provides a recombinant adenovirus comprising coat proteins that lack native binding. In particular, the present invention provides a recombinant adenovirus comprising a penton base protein and a fiber protein, wherein the penton base protein and the fiber protein lack native binding. The present invention further provides a recombinant adenovirus comprising (a) a penton base protein that lacks native binding and (b) a nonnative amino acid sequence that binds a cell-surface binding site.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/101,751, filed Jul. 15, 1998 now U.S. Pat. No. 6,465,253,which, in turn, is the national phase of International PatentApplication PCT/US96/19150, designating the U.S. and filed Nov. 27,1996, a continuation-in-part of U.S. patent application Ser. No.08/563,368, filed Nov. 28, 1995, now U.S. Pat. No. 5,965,541, and acontinuation-in-part of U.S. patent application Ser. No. 08/701,124,filed Aug. 21, 1996, now U.S. Pat. No. 5,846,782, and acontinuation-in-part of U.S. patent application Ser. No. 08/700,846,filed Aug. 21, 1996, now U.S. Pat. No. 5,962,311, which, in turn, is acontinuation-in-part of U.S. patent application Ser. No. 08/634,060,filed Apr. 17, 1996, now U.S. Pat. No. 5,712,136, which, in turn, is acontinuation-in-part of U.S. patent application Ser. No. 08/303,162,filed Sep. 8, 1994, now U.S. Pat. No. 5,559,099.

TECHNICAL FIELD OF THE INVENTION

The present invention pertains to a chimeric adenovirus coat proteinwhich is able to direct entry into cells of a vector comprising the coatprotein that is more efficient than a similar vector having a wild-typeadenovirus coat protein. Such a chimeric coat protein is a fiber, hexon,or penton protein. The present invention also pertains to a recombinantvector comprising such a chimeric adenoviral coat protein, and tomethods of constructing and using such a vector.

BACKGROUND OF THE INVENTION

Adenoviruses belong to the family Adenoviridae, which is divided intotwo genera, namely Mastadenovirus and Aviadenovirus. Adenoviruses arenonenveloped, regular icosahedrons of about 65 to 80 nanometers indiameter (Home et al., J. Mol. Biol., 1, 84-86 (1959)). The adenoviralcapsid is composed of 252 capsomeres of which 240 are hexons and 12 arepentons (Ginsberg et al., Virology, 28, 782-783 (1966)). The hexons andpentons are derived from three different viral polypeptides (Maizel etal., Virology, 36, 115-125 (1968); Weber et al., Virology, 76, 709-724(1977)). The hexon comprises three identical polypeptides of 967 aminoacids each, namely polypeptide II (Roberts et al., Science, 232,1148-1151 (1986)). The penton contains a penton base, which is bound tothe capsid, and a fiber, which is noncovalently bound to and projectsfrom the penton base. The fiber protein comprises three identicalpolypeptides of 582 amino acids each, namely polypeptide IV. Theadenovirus serotype 2 (Ad2) penton base protein is a ring-shaped complexcomposed of five identical protein subunits of 571 amino acids each,namely polypeptide III (Boudin et al., Virology, 92, 125-138 (1979)).Proteins IX, VI, and IIIa are also present in the adenoviral coat andare thought to stabilize the viral capsid (Stewart et al., Cell, 67,145-154 (1991); Stewart et al., EMBO J., 12(7), 2589-2599 (1993)).

Once an adenovirus attaches to a cell, it undergoes receptor-mediatedinternalization into clathrin-coated endocytic vesicles of the cell(Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al.,Virology, 40, 462-477 (1970)). Virions entering the cell undergo astepwise disassembly in which many of the viral structural proteins areshed (Greber et al., Cell, 75, 477-486 (1993)). During the uncoatingprocess, the viral particles cause disruption of the cell endosome by apH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)),which is still poorly understood. The viral particles are thentransported to the nuclear pore complex of the cell (Dales et al.,Virology, 56, 465-483 (1973)), where the viral genome enters thenucleus, thus initiating infection.

An adenovirus uses two separate cellular receptors, both of which mustbe present, to efficiently attach to and infect a cell (Wickham et al.,Cell, 73, 309-319 (1993)). First, the Ad2 fiber protein attaches thevirus to a cell by binding to an as yet unidentified receptor. Then, thepenton base binds to α_(v) integrins, which are a family ofheterodimeric cell-surface receptors that mediate cellular adhesion tothe extracellular matrix molecules, as well as other molecules (Hynes,Cell, 69, 11-25 (1992)).

The fiber protein is a trimer (Devaux et al., J. Molec. Biol., 215,567-588 (1990)) consisting of a tail, a shaft, and a knob. The fibershaft region is composed of repeating 15 amino acid motifs, which arebelieved to form two alternating β-strands and β-bends (Green et al.,EMBO J., 2, 1357-1365 (1983)). The overall length of the fiber shaftregion and the number of 15 amino-acid repeats differ between adenoviralserotypes. For example, the Ad2 fiber shaft is 37 nanometers long andcontains 22 repeats, whereas the Ad3 fiber is 11 nanometers long andcontains 6 repeats. The receptor binding domain of the fiber protein islocalized in the knob region encoded by the last 200 amino acids of theprotein (Henry et al., J. Virology, 68(8), 5239-5246 (1994)). Theregions necessary for trimerization are also located in the knob regionof the protein (Henry et al. (1994), supra). A deletion mutant lackingthe last 40 amino acids does not trimerize and also does not bind topenton base (Novelli et al., Virology, 185, 365-376 (1991)). Thus,trimerization of the fiber protein is necessary for penton base binding.Nuclear localization signals that direct the protein to the nucleus toform viral particles following its synthesis in the cytoplasm arelocated in the N-terminal region of the protein (Novelli et al. (1991),supra). The fiber, together with the hexon, are the main antigenicdeterminants of the virus and also determine the serotype specificity ofthe virus (Watson et al., J. Gen. Virol., 69, 525-535 (1988)).

Recombinant adenoviral vectors have been used for the cell-targetedtransfer of one or more recombinant genes to diseased cells or tissue inneed of treatment. Such vectors are characterized by the advantage ofnot requiring host cell proliferation for expression of adenoviralproteins (Horwitz et al., In Virology, Raven Press, New York, vol. 2,pp. 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)).Moreover, if the targeted tissue for somatic gene therapy is the lung,these vectors have the added advantage of being normally trophic for therespiratory epithelium (Straus, In Adenoviruses, Plenum Press, New York,pp. 451-496 (1984)).

Other advantages of adenoviruses as potential vectors for human genetherapy are: (i) recombination is rarely observed with use of suchvectors; (ii) there are no known associations of human malignancies withadenoviral infections despite common human infection with adenoviruses;(iii) the adenoviral genome (which is a linear, double-stranded DNA) canbe manipulated to accommodate foreign genes that range in size; (iv) anadenoviral vector does not insert its DNA into the chromosome of a cell,so its effect is impermanent and unlikely to interfere with the cell'snormal function; (v) the adenovirus can infect non-dividing orterminally differentiated cells, such as cells in the brain and lungs;and (vi) live adenovirus, having as an essential characteristic theability to replicate, has been safely used as a human vaccine (Horwitzet al. (1990), sura; Berkner et al. (1988), supra; Straus et al. (1984),supra; Chanock et al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J.Virol., 57, 267 (1986); and Ballay et al., EMBO, 4, 3861 (1985); PCTpatent application WO 94/17832).

A drawback to adenovirus-mediated gene therapy is that significantdecreases in gene expression are observed after two weeks followingadministration of the vector. In many therapeutic applications, the lossof expression requires re-administration of the viral vector. However,following re-administration, neutralizing antibodies are raised againstboth the fiber and hexon proteins of the viral vector (Wohlfart, J.Virology, 62, 2321-2328 (1988); Wohlfart et al., J. Virology, 56,896-903 (1985)). This antibody response against the virus can preventeffective re-administration of the viral vector.

Another drawback of using recombinant adenovirus in gene therapy is thatcertain cells are not readily amenable to adenovirus-mediated genedelivery. For instance, lymphocytes, which lack the α_(v) integrinadenoviral receptors, are impaired in the uptake of adenoviruses (Silveret al., Virology 165, 377-387 (1988); Horvath et al., J. Virology,62(1), 341-345 (1988)). This lack of ability to infect all cells haslead researchers to seek out ways to introduce adenovirus into cellsthat cannot be infected by adenovirus, e.g. due to lack of adenoviralreceptors. In particular, the virus can be coupled to a DNA-polylysinecomplex containing a ligand (e.g., transferrin) for mammalian cells(e.g., Wagner et al., Proc. Natl. Acad. Sci., 89, 6099-6103 (1992); PCTpatent application WO 95/26412). Similarly, adenoviral fiber protein canbe sterically blocked with antibodies, and tissue-specific antibodiescan be chemically linked to the viral particle (Cotten et al., Proc.Natl. Acad. Sci. USA, 89, 6094-6098 (1992)).

However, these approaches are disadvantageous in that they requireadditional steps that covalently link large molecules, such aspolylysine, receptor ligands, and antibodies, to the virus (Cotten(1992), supra; Wagner et al., Proc. Natl. Acad. Sci., 89, 6099-6103(1992)). This adds to the size of the resultant vector as well as itscost of production. Moreover, the targeted particle complexes are nothomogeneous in structure, and their efficiency is sensitive to therelative ratios of viral particles, linking molecules, and targetingmolecules used. Thus, this approach for expanding the repertoire ofcells amenable to adenoviral-mediated gene therapy is less than optimal.

Recently, the efficiency of adenovirus-mediated gene transfer in vivo toeven those cells which adenovirus has been reputed to enter with highefficiency has been called into question (Grubb et al., Nature, 371,802-806 (1994); Dupuit et al., Human Gene Therapy, 6, 1185-1193 (1995)).The fiber receptor by means of which adenovirus initially contacts cellshas not been identified, and its representation in different tissues hasnot been examined. It is generally assumed that epithelial cells in thelung and gut produce sufficient levels of the fiber receptor to allowtheir optimal transduction. However, no studies have confirmed thispoint to date. In fact, studies have suggested that adenovirus genedelivery to differentiated lung epithelium is less efficient thandelivery to proliferating or to undifferentiated cells (Grubb et al.,supra; Dupuit et al., supra).

Similarly, adenovirus has been shown to transduce a large number oftissues including lung epithelial cells (Rosenfeld et al., Cell, 68,143-155 (1992)), muscle cells (Quantin et al., Proc. Natl. Acad. Sci.,89, 2581-2584 (1992)), endothelial cells (Lemarchand et al, Proc. Natl.Acad. Sci., 89, 6482-6486 (1992), fibroblasts (Anton et al., J. Virol.,69, 4600-4606 (1995), and neuronal cells (LaSalle et al., Science, 259,988-990 (1993)). However, in many of these studies, very high levels ofvirus particles have been used to achieve transduction, often exceeding100 plaque forming units (pfu)/cell, and corresponding to a multiplicityof infection (MOI) of 100. The requirement for a high MOI to achievetransduction is disadvantageous inasmuch as any immune responseassociated with adenoviral infection necessarily would be exacerbatedwith use of high doses.

Accordingly, there remains a need for vectors, such as adenoviralvectors, that are capable of infecting cells with a high efficiency,especially at lower MOIs, and that demonstrate an increased host cellrange of infectivity. The present invention seeks to overcome at leastsome of the aforesaid problems of recombinant adenoviral gene therapy.In particular, it is an object of the present invention to provide avector (such as an adenoviral vector) having a broad host range, and anability to enter cells with a high efficiency, even at a reduced MOI,thereby reducing the amount of recombinant adenoviral vectoradministered and any side-effects/complications resulting from suchadministration. A further object of the present invention is to providea method of gene therapy involving the use of a homogeneous adenovirus,wherein the viral particle is modified at the level of the adenoviralgenome, without the need for additional chemical modifications of viralparticles. These and other objects and advantages of the presentinvention, as well as additional inventive features, will be apparentfrom the following detailed description.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a chimeric adenoviral coat protein (e.g.,a fiber, hexon or penton protein), which differs from the wild-type(i.e., native) fiber protein by the introduction of a nonnative aminoacid sequence, preferably at or near the carboxyl terminus. Theresultant chimeric adenovirus coat protein is able to direct entry intocells of a vector comprising the coat protein that is more efficientthan entry into cells of a vector that is identical except forcomprising a,wild-type adenovirus coat protein rather than the chimericadenovirus coat protein. One direct result of this increased efficiencyof entry is that the chimeric adenovirus coat protein enables theadenovirus to bind to and enter numerous cell types which adenoviruscomprising wild-type coat protein typically cannot enter or can enterwith only a low efficiency. The present invention also provides anadenoviral vector that comprises the chimeric adenovirus coat protein,and methods of constructing and using such a vector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph depicting the binding (percent of input) ofwild-type adenovirus to cells derived from different tissues.

FIGS. 2A-B depict attachment of a nucleic acid sequence at the end ofthe wild-type adenoviral fiber gene (FIG. 2A) to derive a chimericadenoviral fiber protein (FIG. 2B) comprising a nonnative amino acidsequence at the carboxy terminus. As indicated, the length of the polyAtail, and, consequently, the number of lysines in the resultant protein,can vary.

FIG. 3 is a schematic diagram depicting the construction of theadenovirus transfer vector containing chimeric fiber protein pAd BS59-100 UTV by way of intermediary transfer vectors. In particular, pAdNS 83-100 (also known as p193NS 83-100 or pNS 83-100) is used to derivefiber minus (i.e., F⁻) pAd NS 83-100 (also known as p193NS (ΔF) or pNS(ΔF)) (path A), pAdNS 83 100 (F⁻) is used to derive pAd NS 83-100 UTV(also known as p193NS (F5*), p193 (F5*), or pNS (F5*)) (path B), and pAdNS 83-100 UTV is used to derive pAd BS 59-100 UTV (path C).

FIGS. 4A-D depict the oligonucleotides employed for construction of GV10UTV, i.e., the primers SEQ ID NO:9 (FIG. 4A), SEQ ID NO:10 (FIG. 4B),SEQ ID NO:11 (FIG. 4C), and SEQ ID NO:12 (FIG. 4D).

FIG. 5 depicts a Western blot showing the size increase of the chimericadenoviral fiber protein (UTV) as compared with the wild-type fiberprotein (WT).

FIGS. 6A-B are graphs depicting a comparison of the binding of anadenoviral vector comprising wild-type fiber protein (i.e., GV10, opentriangle) and adenoviral vector comprising chimeric fiber protein (i.e.,GV10 UTV, filled circle) to a receptor-plus (A549, FIG. 6A) and areceptor-minus (HS 68, FIG. 6B) cell.

FIG. 7 is a graph of UTV bound (counts per minute (CPM)) versus amountof competitor (μg/ml) for inhibition of binding of chimeric adenoviralfiber protein to receptor-minus cells (i.e., HS 68 fibroblasts) by thesoluble factors chondroitin sulfate (open circle); heparin (filledcircle); mucin (filled triangle); and salmon sperm DNA (open triangle).

FIG. 8 is a graph of UTV bound (CPM) versus enzyme dilution forinhibition of binding of chimeric adenoviral fiber protein toreceptor-minus cells (i.e., HS 68 fibroblasts) by the enzymeschondroitinase (open circles, stippled lines); heparinase (open circles,solid lines); and sialidase (triangles, solid lines).

FIG. 9 is a bar graph depicting a comparison of transfer of a lacZreporter gene by an adenoviral vector comprising wild-type fiber protein(i.e., GV10) and an adenoviral vector comprising chimeric fiber protein(i.e., GV10 UTV) as assessed by resultant reporter gene expression(i.e., relative light units (RLU)) in various receptor-plus andreceptor-minus cells.

FIG. 10 is a bar graph depicting a comparison of transfer of a lacZreporter gene by an adenoviral vector comprising wild-type fiber protein(i.e., GV10) and an adenoviral vector comprising chimeric fiber protein(i.e., GV10 UTV) as assessed by resultant reporter expression (i.e.,relative light units (RLU)) in mouse lung.

FIG. 11 is a bar graph depicting the transfer of a reporter gene (i.e.,contained in pGUS) by an adenoviral vector comprising wild-type fiberprotein (i.e., GV10, solid bars) and an adenoviral vector comprisingchimeric fiber (i.e., GV10 UTV, open bars) potentially bound via aprotein/DNA interaction into 293 cells, A549 cells, and H700 T cells.

FIG. 12 is a diagram that further depicts the plasmid p193 (F5*)(described as pAd NS 83-100 UTV in FIG. 3, and also known as p193 (F5*)or pNS (F5*)) used to construct adenovirus fiber chimeras, and thesequence of the C-terminus of the mutated fiber protein present in theplasmid (polyadenylation site emboldened).

FIG. 13 is a diagram that depicts plasmid p193NS (F5*) pGS(K7) (alsoknown as p193 (F5*) pGS(K7) or pNS (F5*) pK7) used to constructadenovirus fiber chimeras.

FIG. 14 is a diagram that depicts plasmid pBSS 75-100 pGS(null) (alsoknown as pBSS 75-100 ΔE3 pGS(null)).

FIG. 15 is a diagram that depicts plasmid pBSS 75-100 pGS(RK32) (alsoknown as pBSS 75-100 ΔE3 pGS(RKKK)₂ or pBSS 75-100 ΔE3 pGS(RKKK2)).

FIG. 16 is a diagram that depicts plasmid pBSS 75-100 pGS(RK33) (alsoknown as pBSS 75-100 ΔE3 pGS(RKKK)₃ or pBSS 75-100 ΔE3 pGS(RKKK3)).

FIG. 17 is a diagram that depicts plasmid p193NS F5F2K (also known asp193 F5F2K).

FIG. 18 is a diagram that depicts plasmid p193NS F5F2K(RKKK)₂ (alsoknown as p193NS F5F2K(RKKK2), p193NS F5F2K(RK32), or p193 F5F2K(RKKK2)).

FIG. 19 is a diagram that depicts plasmid p193NS F5F2K(RKKK)₃ (alsoknown as p193NS F5F2K(RKKK3), p193 F5F2K(RKKK3), or p193 F5FK(RK33)).

FIG. 20 is a diagram that depicts plasmid pACT (RKKK)₃ (also known aspACT (RKKK3) or pACT (RK33)).

FIG. 21 is a diagram that depicts plasmid pACT (RKKK)₂ (also known aspACT (RKKK2) or pACT (RK32)).

FIG. 22 is a diagram that depicts plasmid pACT H11.

FIG. 23 is a diagram that depicts plasmid pACT H11(RKKK)₂ (also known aspACT H11 (RKKK2) or pACT H11(RK32)).

FIG. 24 is a diagram that depicts plasmid p193 F5F9sK (also known asp193 F5F9K-Short).

FIG. 25 is a diagram that depicts plasmid pSPdelta.

FIG. 26 is a diagram that depicts plasmid pSP2alpha. The “j” indicatesdestroyed Ppu10I sites in the plasmid.

FIG. 27 is a diagram that depicts plasmid pSP2alpha2. The “j” indicatesdestroyed Ppu10I sites in the plasmid.

FIG. 28 is a graph of days post-infection versus FFU/cell for 293 cellsinfected with Ad5 (open circles). AdZ.F(RGD) (closed squares), orAdZ.F(pK7) (open triangles).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, among other things, a recombinantadenovirus comprising a chimeric coat protein, such as a chimeric fiber,penton, and/or hexon protein. The chimeric coat protein comprises anormative amino acid sequence, in addition to, or in place of, a nativeamino acid sequence. This normative amino acid sequence allows thechimeric fiber (or a vector comprising the chimeric fiber) to moreefficiently bind to and enter cells.

Thus, the present invention provides, a chimeric adenovirus coat proteincomprising a normative amino acid sequence, wherein the coat protein isable to direct entry into cells of a vector comprising the coat proteinthat is more efficient than entry into cells of a vector that isidentical except for comprising a wild-type adenovirus coat proteinrather than the chimeric adenovirus coat protein (i.e., in the absenceof the chimeric adenovirus coat protein and in the presence of thewild-type adenovirus coat protein).

Chimeric Coat Protein

A “coat protein” according to the invention preferably comprises a fiberprotein (especially an adenoviral fiber protein), a penton protein(especially an adenoviral penton protein), and a hexon protein(especially an adenoviral hexon protein). In particular, a coat proteinpreferably comprises an adenoviral fiber, penton, or hexon protein. Anyone of the serotypes of human or nonhuman adenovirus can be used as thesource of the coat protein gene, optimally, however, the adenovirus isan Ad2 or Ad5 adenovirus.

The coat protein is “chimeric” in that it comprises amino acid residuesthat are not typically found in the protein as isolated from wild-typeadenovirus (i.e., comprising the native protein, or wild-type protein).The coat protein thus comprises a “normative amino acid sequence”. By“nonnative amino acid sequence” is meant any amino acid sequence that isnot found in the native fiber of a given serotype of adenovirus andwhich preferably is introduced into the fiber protein at the level ofgene expression (i.e., by introduction of a “nucleic acid sequence thatencodes a normative amino acid sequence”).

Such a normative amino acid sequence comprises an amino acid sequence(i.e., has component residues in a particular order) which imparts uponthe resultant chimeric protein an ability to bind to and enter cells bymeans of a novel cell surface binding site (i.e., a “UTV sequence”, orUniversal Transfer Vector sequence), and/or comprises a sequenceincorporated to produce or maintain a certain configuration of theresultant chimeric protein (i.e., a “spacer sequence”) betweennative/nonnative, nonnative/normative, or a native/native sequence.Inasmuch as the normative amino acid sequence is inserted into or inplace of an amino acid sequence, and the manipulation of the amino acidsequence of the chimeric coat protein preferably is made at the nucleicacid level, the amino acid sequence that differs in the chimeric coatprotein from the wild-type coat protein (i.e., the UTV sequence and thespacer sequence) preferably can comprise an entirely normative aminoacid sequence, or a mixture of native and normative amino acids).

A “cell surface binding site” encompasses a receptor (which preferablyis a protein, carbohydrate, glycoprotein, or proteoglycan) as well asany oppositely charged molecule (i.e., oppositely charged with respectto the chimeric coat protein, which preferably comprises a normativeamino acid sequence that is positively charged, as described furtherherein) or other type of molecule with which the chimeric coat proteincan interact to bind the cell, and thereby promote cell entry. Examplesof potential cell surface binding sites include, but are not limited to:negatively charged heparin, heparan sulfate, hyaluronic acid, dermatansulfate, and chondroitin sulfate moieties, for instance, found onglycosaminoglycans, and which further may contain sialic acid, sulfate,and/or phosphate; sialic acid moieties found on mucins, glycoproteins,and gangliosides; major histocompatibility complex I (MHC I)glycoproteins; common carbohydrate components found in membraneglycoproteins, including mannose, N-acetyl-galactosamine,N-acetyl-glucosamine, fucose, galactose, and the like; and phosphatemoieties, for instance, on nucleic acids. However, a chimeric coatprotein according to the invention, and methods of use thereof, is notlimited to any particular mechanism of cellular interaction (i.e.,interaction with a particular cell surface binding site) and is not tobe so construed.

Furthermore, such a cell surface binding site is “novel” inasmuch as thesite is one that previously was inaccessible to interaction with anadenoviral coat protein (i.e., wild-type adenoviral coat protein such asfiber protein), or was accessible only at a very low level, as reflectedby the reduced efficiency of entry of a wild-type adenoviral coatprotein-containing vector as compared with a vector comprising achimeric adenovirus coat protein such as fiber protein according to theinvention. Moreover, the binding site is novel in that it is present onthe majority of, if not all, cells, regardless of their origin. This isin contrast to the cellular binding site with which wild-type adenoviralfiber protein is presumed to interact, which ostensibly is present onlyon a subset of cells, or is only accessible on a subset of cells, asreflected by the reduced efficiency of entry of a wild-type adenoviralfiber-containing vector.

“Efficiency of entry” can be quantitated by several means. Inparticular, efficiency of entry can be quantitated by introducing achimeric coat protein into a vector, preferably a viral vector, andmonitoring cell entry (e.g., by vector-mediated delivery to a cell of agene such as a reporter gene) as a function of multiplicity of infection(MOI)). In this case, a reduced MOI required for cell entry of a vectorcomprising a chimeric adenoviral coat protein as compared with a vectorthat is identical except for comprising a wild-type adenoviral coatprotein rather than said chimeric adenovirus coat protein, indicates“more efficient” entry.

Similarly, efficiency of entry can be quantitated in terms of theability of vectors containing chimeric or wild-type coat proteins, orthe soluble chimeric or wild-type coat proteins themselves, to bind tocells. In this case, increased binding exhibited for the vectorcontaining a chimeric adenoviral coat protein, or the chimeric coatprotein itself, as compared with the identical vector containing awild-type coat protein instead, or the wild-type coat protein itself, isindicative of an increased efficiency of entry, or “more efficient”entry.

A nonnative amino acid sequence according to the invention preferably isinserted into or in place of an internal coat protein sequence.Alternately, preferably a normative amino acid sequence according to theinvention is at or near the C-terminus of a protein. In particular, whena coat protein according to the invention is a fiber protein, desirablya normative amino acid sequence is at or near the C-terminus of theprotein. When a coat protein according to the invention is a penton orhexon protein, preferably a normative amino acid sequence is within anexposed loop of the protein, e.g., as described in the followingExamples, particularly within a hypervariable region in loop 1 and/orloop 2 of the adenovirus hexon protein (Crawford-Miksza et al., J.Virol., 70, 1836-1844 (1996)). Thus, desirably a nonnative amino acidsequence is in a region of a coat protein that is capable of interactingwith and binding a cell.

Furthermore, the method of the invention can be employed to createadenoviral vectors that contain UTV or UTV-like sequences in an extended(or spiked) structure, particularly in hexon and/or penton base protein,so as to result in lengthened hexon and/or penton base proteins, whereinthe amino acid insertion projects outward from the protein as it ispresent in a virion capsid. In particular, such spiked coat proteins canbe incorporated into a recombinant adenovirus along with a“short-shafted fiber” (further described herein), wherein the shaft ofthe fiber has been shortened, and, optionally, the knob of the fiberprotein has been replaced with a knob (including the trimerizationdomain) of another serotype adenoviral vector from which the remainderof the fiber protein derived.

Accordingly, the short-shafted fiber protein preferably can beincorporated into an adenovirus having a chimeric penton base proteinthat comprises a UTV or UTV-like sequence, or having a “spiked” chimericpenton base protein that furthermore optionally can incorporate a UTV orUTV-like sequence. Also, the short-shafted fiber protein can beincorporated into an adenovirus having a chimeric hexon protein thatcomprises a UTV or UTV-like sequence, or having a “spiked” chimerichexon protein that furthermore optionally can incorporate a UTV orUTV-like sequence.

Optimally, the nonnative amino acid sequence is linked to the protein byanother normative amino acid sequence, i.e., by an intervening spacersequence. A spacer sequence is a sequence that intervenes between thenative protein sequence and the nonnative sequence, between a normativesequence and another normative sequence, or between a native sequenceand another native sequence. A spacer sequence preferably isincorporated into the protein to ensure that the normative sequencecomprising the cell surface binding site projects from the threedimensional structure of the chimeric protein (especially the threedimensional structure of the chimeric protein as it exists in nature,i.e., as part of a capsid) in such a fashion so as to be able tointeract with and bind to cells. When a spacer sequence is inserted intoor replaces an internal coat protein sequence, one or more spacersequences may be present in the chimeric coat protein.

An intervening spacer sequence can be of any suitable length, preferablyfrom about 3 to about 400 amino acids for a spacer sequence added toderive a “spiked” coat protein (as further described in the followingExamples), and preferably from about 3 to about 30 amino acids for anyother application described herein. A spacer sequence can comprise anyamino acids. Optimally, the spacer sequence does not interfere with thefunctioning of the coat protein in general, and the functioning of theother normative amino acid sequence (i.e., the UTV or UTV-like sequence)in particular.

The nonnative amino acid sequence which is not a spacer sequence, i.e.,the UTV sequence, also can be of any suitable length, preferably fromabout 3 to about 30 amino acids (although, optionally, as for the spacersequence, the UTV sequence can be longer, e.g., up to about 400 aminoacids). These amino acids preferably are any positively charged residuesthat are capable of binding to negatively charged moieties present onthe surface of a eukaryotic cell, and optimally are capable of bindingto negatively charged moieties that are present on the surface of themajority of (if not all) eukaryotic cells. In particular, such anegatively charged moiety present on the surface of a eukaryotic cell towhich the UTV sequence binds includes the aforementioned “cell surfacebinding site”.

Desirably the normative amino acid sequence comprises amino acidsselected from the group consisting of lysine, arginine and histidine.Alternately, these amino acids can be negatively charged residues thatare capable of binding to positively charged cell surface binding sites,e.g., desirably the normative amino acid sequence comprises amino acidsselected from the group consisting of aspartate and glutamate.

Thus, the normative amino acid sequence of a coat protein preferablycomprises a sequence selected from the group consisting of SEQ ID NO:1(i.e., Lys Lys Lys Lys Lys Lys Lys Lys), SEQ ID NO:2 (i.e., Arg Arg ArgArg Arg Arg Arg Arg), and SEQ ID NO:3 (i.e., Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa, wherein “Xaa” comprises Lys or Arg), and wherein 1, 2, 3, 4, or 5residues of the sequence may be deleted at the C-terminus thereof. Whenthe coat protein is a fiber protein, preferably the protein comprises asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 (i.e., Gly Ser Asn Lys Glu Ser Phe Val Leu LysLys Lys Lys Lys Lys), and SEQ ID NO:5 (i.e., Ala Gly Ser Asn Lys Asn LysGlu Ser Phe Val Leu Lys Lys Lys Lys Lys Lys), and wherein 1, 2, 3, 4, or5 residues of the sequence may be deleted at the C-terminus thereof.

Also, sequences that bind that bind to heparin may be involved inbinding to a heparin-like receptor (Sawitzky et al., Med. Microbiol.Immunol., 182, 285-92 (1993). Similarly, so-called “heparin bindingsequences” may mediate the interaction of the peptide or protein inwhich they are contained with other cell surface binding sites, such aswith cell surface heparan sulfate proteoglycan (Thompson et al., J.Biol. Chem., 269, 2541-9 (1994)). Thus, preferably the normative aminoacid sequence (i.e., the UTV sequence) comprises these sequences, aswell as additional sequences that are capable of recognizing anegatively charged moiety broadly represented on the surface ofeukaryotic cells.

In particular, preferably the normative amino acid sequence comprisestwo basic amino acids (frequently Arg) located about 20 Å apart, facingin opposite directions of an alpha helix (Margalit et al., J. Biol.Chem., 269, 19228-31 (1993); Ma et al., J. Lipid Res., 35, 2049-2059(1994)). Other basic amino acids desirably are dispersed between thesetwo residues, facing one side, while nonpolar residues face the otherside, forming an amphipathic structure, which optimally comprises themotif XBBXBX [SEQ ID NO:49] or XBBBXXBX [SEQ ID NO:50], where B is abasic amino acid (e.g., Lys, Arg, etc.), and X is any other amino acid.

Also, preferably the UTV nonnative amino acid sequence comprises: thesequence LIGRKKT [SEQ ID NO:51], LIGRK [SEQ ID NO:52] or LIGRR [SEQ IDNO:53], which are common heparin binding motifs present in fibronectinand heat shock proteins (Hansen et al., Biochim. Biophys. Acta, 1252,135-45 (1995)); insertions of 7 residues of either Lys or Arg, ormixtures of Lys and Arg (Fromm et al., Arch. Biochem. Biophys., 323,279-87 (1995)); the common basic C-terminal region of IGFBP-3 andIGFBP-5 of about 18 amino acids and which comprises a heparin bindingsequence (Booth et al., Growth Regul., 5, 1-17 (1995)); either one orboth of the two hyaluronan (HA) binding motifs located within a 35 aminoacid region of the C-terminus of the HA receptor RHAMM (Yang et al., J.Cell Biochem., 56, 455-68 (1994)); a synthetic peptide (Ala347-Arg361)modeled after the heparin-binding form of Staphylococcus aureusvitronectin comprising heparin-binding consensus sequences (Liang etal., J. Biochem., 116, 457-63 (1994)); any one or more of five heparinbinding sites between amino acid 129 and 310 of bovine herpesvirus 1glycoprotein gIII or any one of four heparin binding sites between aminoacids 90 and 275 of pseudorabies virus glycoprotein gIII (Liang et al.,Virol., 194, 233-43 (1993)); amino acids 134 to 141 of pseudorabiesvirus glycoprotein gIII (Sawitzky et al., Med. Microbiol. Immunol., 182,285-92 (1993); heparin binding regions corresponding to charged residuesat positions 279-282 and 292-304 of human lipoprotein lipase (Ma et al.,supra); a synthetic 22 residue peptide, N22W, with a sequenceNVSPPRRARVTDATETTITISW [SEQ ID NO:54] or residues TETTITIS [SEQ IDNO:55] of this synthetic peptide modeled after fibronectin and whichexhibit heparin binding properties (Ingham et al., Arch. Biochem.Biophys., 314, 242-246 (1994)); GVEFVCCP [SEQ ID NO:56] motif present inthe ectodomain zinc binding site of the Alzheimer beta-amyloid precursorprotein (APP), as well as various other APP-like proteins, whichmodulates heparin affinity (Bush et al., J. Biol. Chem., 229, 26618-21(1994)); 8 amino acid residue peptides derived from the cross-region ofthe laminin A chain (Tashiro et al., Biochem. J., 302, 73-9 (1994));synthetic peptides based on the heparin binding regions of the serineprotease inhibitor antithrombin III including peptides F123-G148 andK121-A134 (Tyler-Cross et al., Protein Sci., 3, 620-7 (1994)); a 14 KN-terminal fragment of APP and a region close to the N-terminus (i.e.,residues 96-110) proposed as heparin binding regions (Small et al., J.Neurosci., 14, 2117-27 (1994)); a stretch of 21 amino acids of theheparin binding epidermal growth factor-like growth factor (HB-EGF)characterized by a high content of lysine and arginine residues(Thompson et al., J. Biol. Chem., 269, 2541-9 (1994)); a 17 amino acidregion comprising the heparin binding region of thrombospondin andcorresponding to a hep 1 synthetic peptide (Murphy-Ullrich et al., J.Biol. Chem., 268, 26784-9 (1993)); a 23 amino acid sequence (Y565-A587)of human von Willebrand factor that binds heparin (Tyler-Cross et al.,Arch. Biochem. Biophys., 306, 528-33 (1993)); the fibronectin-derivedpeptide PRARI [SEQ ID NO:57](and larger peptides comprising this motif,such as WQPPRARI [SEQ ID NO:58]) that binds heparin (Woods et al., Mol.Biol. Cell., 4, 605-613 (1993); the heparin binding region of plateletfactor 4 (Sato et al., Jpn. J. Cancer Res., 84, 485-8 (1993); and theK18K sequence in the fibroblast growth factor receptor tyrosine kinasetransmembrane glycoprotein (Kan et al., Science, 259, 1918-21 (1993)).

Moreover, the UTV sequence can comprise other sequences that aredescribed in the Examples which follow. Thus, preferably the UTVsequence is selected from the group consisting of [SEQ ID NO:19 , [SEQID NO:2], [SEQ ID NO:3], [SEQ ID NO:4], [SEQ ID NO:5], [SEQ ID NO:20],[SEQ ID NO:22], [SEQ ID NO:24], [SEQ ID NO:26], [SEQ ID NO:28], [SEQ IDNO:30], [SEQ ID NO:32],[SEQ ID NO:34], [SEQ ID NO:36], [SEQ ID NO:38],[SEQ ID NO:40], [SEQ ID NO:42], [SEQ ID NO:46], [SEQ ID NO:48], [SEQ IDNO:49], [SEQ ID NO:50], [SEQ ID NO:5], [SEQ ID NO:52], [SEQ ID NO:53],[SEQ ID NO:54], [SEQ ID NO:55], [SEQ ID NO:56], [SEQ ID NO:57], [SEQ IDNO:58], [SEQ ID NO:73], [SEQ ID NO:74], [SEQ ID NO:76], [SEQ IDNO:78],[SEQ ID NO:90], and [SEQ ID NO:93]. These sequences also can beemployed wherein 1, 2, or 3 residues of the sequence are deleted at theC- or N-terminus. Also, inasmuch as a spacer sequence can be anysequence of amino acids that does not interfere with the functioning ofthe protein, according to the invention, any of the aforementioned UTVsequences also can comprise spacer sequences.

It also is preferable that the nonnative amino acid sequence compriseamino acid sequences that are “equivalents” of any of the aforementionedsequences (i.e., are “UTV-like sequences”). An equivalent can be asequence that carries out the same function (with perhaps minordifferences in effectiveness), and yet may differ slightly in terms ofits amino acid sequence, or other structural features. In particular, anequivalent sequence is one that comprises one or more conservative aminoacid substitutions of the sequence. A “conservative amino acidsubstitution” is an amino acid substituted by an alternative amino acidof similar charge density, hydrophilicity/hydrophobicity, size, and/orconfiguration (e.g., Val for I1e). In comparison, a “nonconservativeamino acid substitution” is an amino acid substituted by an alternativeamino acid of differing charge density, hydrophilicity/hydrophobicity,size, and/or configuration (e.g., Val for Phe).

Nucleic Acid Encoding a Chimeric Coat Protein

As indicated previously, preferably the normative amino acid sequence isintroduced at the level of DNA. Accordingly, the invention preferablyalso provides an isolated and purified nucleic acid encoding a coatprotein according to the invention, wherein the nucleic acid sequencethat encodes the nonnative amino acid sequence comprises a sequence ofSEQ ID NO:6 (i.e., GGA TCC AA), which is located prior to thepolyadenylation site. Similarly, the invention preferably provides anisolated and purified nucleic acid comprising a sequence selected fromthe group consisting of SEQ ID NO:7 (i.e., GGA TCC AAT AAA GAA TCG TTTGTG TTA TGT) and SEQ ID NO:8 (i.e., GCC GGA TCC AAC AAG AAT AAA GAA TCGTTT GTG TTA), [SEQ ID NO:19], [SEQ ID NO:21], [SEQ ID NO:23], [SEQ IDNO:25], [SEQ ID NO:27], [SEQ ID NO:29], [SEQ ID NO:31], [SEQ ID NO:33],[SEQ ID NO:35], [SEQ ID NO:37], [SEQ ID NO:39], [SEQ ID NO:41], [SEQ IDNO:45], [SEQ ID NO:47], [SEQ ID NO:72], [SEQ ID NO:75], [SEQ ID NO:77],and [SEQ ID NO:89]. The invention further provides conservativelymodified variants of these nucleic acids.

A “conservatively modified variant” is a variation on the nucleic acidsequence that results in a conservative amino acid substitution. Incomparison, a “nonconservatively modified variant” is a variation on thenucleic acid sequence that results in a nonconservative amino acidsubstitution. The means of making such modifications are well known inthe art, are described in the Examples which follow, and also can beaccomplished by means of commercially available kits and vectors (e.g.,New England Biolabs, Inc., Beverly, MA; Clontech, Palo Alto, Calif.).Moreover, the means of assessing such substitutions (e.g., in terms ofeffect on ability to bind and enter cells) are described in the Examplesherein.

The means of making such a chimeric coat protein, particularly the meansof introducing the sequence at the level of DNA, is well known in theart, is illustrated in FIG. 2 for a representative chimeric protein, andis described in the Examples that follow. Briefly, the method comprisesintroducing a sequence (preferably the sequence of SEQ ID NO:6 or aconservatively modified variant thereof) into the sequence of the coatprotein. In one preferred embodiment described in the Examples whichfollow, the introduction is made prior to any stop codon orpolyadenylation signal so as to induce a frameshift mutation into theresultant protein, such that the chimeric protein incorporatesadditional amino acids.

Generally, this can be accomplished by cloning the fiber sequence into aplasmid or some other vector for ease of manipulation of the sequence.Then, restriction sites flanking the sequence at which the frameshiftmutation is to be introduced are identified. A double-stranded syntheticoligonucleotide is created from overlapping synthetic single-strandedsense and antisense oligonucleotides (e.g., from the sense and antisenseoligonucleotides, respectively, TAT GGA GGA TCC AAT AAA GAA TCG TTT GTGTTA TGT TTC AAC GTG TTT ATT TTT C [SEQ ID NO:9] and AAT TGA AAA ATA AACACG TTG AAA CAT AAC ACA AAC GAT TCT TTA TTG GAT CCT CCA [SEQ ID NO:10],as illustrated in FIG. 4) such that the double-stranded oligonucleotideincorporates the restriction sites flanking the target sequence. Theplasmid or other vector is cleaved with the restriction enzymes, and theoligonucleotide sequence having compatible cohesive ends is ligated into the plasmid or other vector, to replace the wild-type DNA. Othermeans of in vitro site-directed mutagenesis such as are known to thoseskilled in the art, and can be accomplished (in particular, using PCR),for instance, by means of commercially available kits, can be used tointroduce the mutated sequence into the coat protein coding sequence.

Once the sequence is introduced into the chimeric coat protein, thenucleic acid fragment encoding the sequence can be isolated, e.g., byPCR amplification using 5′ and 3′ primers. For instance, with respect toa chimeric fiber protein, the fragment can be isolated by PCR using theprimer TCCC CCCGGG TCTAGA TTA GGA TCC TTC TTG GGC AAT GTA TGA [SEQ IDNO:11], and the primer CGT GTA TCC ATA TGA CAC AGA [SEQ ID NO:12], asillustrated in FIG. 4. Use of these primers in this fashion results inan amplified chimeric fiber-containing fragment that is flanked byrestriction sites (i.e., in this case NdeI and BamHI sites) that can beused for convenient subcloning of the fragment. Other means ofgenerating a chimeric coat protein also can be employed.

Thus, the frameshift mutation can be introduced into any part of a coatprotein coding sequence. With respect to SEQ ID NO:6, for instance, thissequence can be placed at the region of the coat protein gene that codesfor the C-terminus of the protein (i.e., can be added immediately priorto the TAA stop codon), or can be placed earlier into the coding region,such as between codons coding for Ala (i.e., A) and Gln (i.e., Q) toproduce the aforementioned coding sequence of SEQ ID NO:8, which encodesa chimeric protein comprising the sequence of SEQ ID NO:5. Similarly,this approach can be employed to introduce a frameshift even earlier inthe coding sequence, e.g., either inserted into or in place of aninternal (i.e., native) coat protein sequence.

Moreover, the double-stranded oligonucleotide can also incorporate afurther restriction site that also can be employed in manipulating thesequence. For instance, the sequence of SEQ ID NO:6 introduced in thevector comprises a modified BamHI site, i.e., the site is “modified” inthat it adds additional nucleotides onto the palindromic recognitionsequence. This sequence also can be synthesized to comprise any otherrestriction site convenient for DNA manipulations. When incorporatedinto the coat protein coding sequence, the sequence not only introducesa frame shift mutation, but also can be used to introduce other codingsequences into the coat protein gene. In particular, the codingsequences introduced in this fashion can comprise codons for lysine,arginine and histidine, or codons for aspartate and glutamate, eitheralone, or in any combination. Furthermore, a new translation stop codoncan follow these codons for the amino acids, allowing a chimeric proteinto be produced that only incorporates a given number of additional aminoacids in the nonnative amino acid sequence. The codons for the aminoacids and the translation stop codon can be introduced into theframeshift mutation-inducing novel restriction site incorporated intothe coat protein by synthesizing oligonucleotides comprising thesesequences that are flanked by the restriction site as previouslydescribed (e.g., that comprise 5′ and 3′ BamHI sites), or by other suchmeans that are known to those skilled in the art.

The size of the DNA used to replace the native receptor binding sequencemay be constrained, for example, by impeded folding of the fiber orimproper assembly of the penton base/fiber complex. DNA encoding theaforementioned amino acid sequences (e.g., lysine, arginine, histidine,aspartate, glutamate, and the like) is preferred for insertion into thefiber gene sequence in which the native receptor binding sequence hasbeen deleted or otherwise mutated. Moreover, other DNA sequences, suchas those that encode amino acids for incorporation into spacersequences, preferably are used to replace the native coat protein codingsequence.

Vector Comprising a Chimeric Coat Protein

A “vector” according to the invention is a vehicle for gene transfer asthat term is understood by those skilled in the art. Four types ofvectors encompassed by the invention are plasmids, phages, viruses, andliposomes. Plasmids, phages, and viruses can be transferred to a cell intheir nucleic acid form, and liposomes can be employed to transfernucleic acids. Hence, the vectors that can be employed for gene transferare referred to herein as “transfer vectors”.

Preferably, a vector according to the invention is a virus, especially avirus selected from the group consisting of nonenveloped viruses, i.e.,nonenveloped RNA or DNA viruses. Also, a virus can be selected from thegroup consisting of enveloped viruses, i.e., enveloped RNA or DNAviruses. Such viruses preferably comprise a coat protein. Desirably, theviral coat protein is one that projects outward from the capsid suchthat it is able to interact with cells. In the case of enveloped RNA orDNA viruses, preferably the coat protein is in fact a lipid envelopeglycoprotein (i.e., a so-called spike or peplomer).

In particular, preferably a vector is a nonenveloped virus (i.e., eithera RNA or DNA virus) from the family Hepadnaviridae, Parvoviridae,Papovaviridae, Adenoviridae, or Picornaviridae. A preferred nonenvelopedvirus according to the invention is a virus of the familyHepadnaviridae, especially of the genus Hepadnavirus. A virus of thefamily Parvoviridae desirably is of the genus Parvovirus (e.g.,parvoviruses of mammals and birds) or Dependovirus (e.g.,adeno-associated viruses (AAVs)). A virus of the family Papovaviridaepreferably is of the subfamily Papillomavirinae (e.g., thepapillomaviruses including, but not limited to, human papillomaviruses(HPV) 1-48) or the subfamily Polyomavirinae (e.g., the polyomavirusesincluding, but not limited to, JC, SV40 and BK virus). A virus of thefamily Adenoviridae desirably is of the genus Mastadenovirus (e.g.,mammalian adenoviruses) or Aviadenovirus (e.g., avian adenoviruses). Avirus of the family Picornaviridae is preferably a hepatitis A virus(HAV), hepatitis B virus (HBV), or a non-A or non-B hepatitis virus.

Similarly, a vector can be an enveloped virus from the familyHerpesviridae or Retroviridae, or can be a Sindbis virus. A preferredenveloped virus according to the invention is a virus of the familyHerpesviridae, especially of the subfamily or genus Alphaherpesvirinae(e.g., the herpes simplex-like viruses), Simplexvirus (e.g., herpessimplex-like viruses), Varicellavirus (e.g., varicella andpseudorabies-like viruses), Betaherpesvirinae (e.g., thecytomegaloviruses), Cytomegalovirus (e.g., the human cytomegaloviruses),Gammaherpesvirinae (e.g., the lymphocyte-associated viruses), andLymphocryptovirus (e.g., EB-like viruses).

Another preferred enveloped virus is a RNA virus of the familyRetroviridae (i.e., preferably is a retrovirus), particularly a virus ofthe genus or subfamily Oncovirinae, Spumavirinae, Spumavirus,Lentivirinae, and Lentivirus. A RNA virus of the subfamily Oncovirinaeis desirably a human T-lymphotropic virus type 1 or 2 (i.e., HTLV-1 orHTLV-2) or bovine leukemia virus (BLV), an avian leukosis-sarcoma virus(e.g., Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), avianerythroblastosis virus (AEV), Rous-associated virus (RAV)-1 to 50,RAV-0), a mammalian C-type virus (e.g., Moloney murine leukemia virus(MuLV), Harvey murine sarcoma virus (HaMSV), Abelson murine leukemiavirus (A-MuLV), AKR-MuLV, feline leukemia virus (FeLV), simian sarcomavirus, reticuloendotheliosis virus (REV), spleen necrosis virus (SNV)),a B-type virus (e.g., mouse mammary tumor virus (MMTV)), or a D-typevirus (e.g., Mason-Pfizer monkey virus (MPMV), “SAIDS” viruses). A RNAvirus of the subfamily Lentivirus is desirably a human immunodeficiencyvirus type 1 or 2 (i.e., HIV-1 or HIV-2, wherein HIV-1 was formerlycalled lymphadenopathy associated virus 3 (HTLV-III) and acquired immunedeficiency syndrome (AIDS)-related virus (ARV)), or another virusrelated to HIV-1 or HIV-2 that has been identified and associated withAIDS or AIDS-like disease. The acronym “HIV” or terms “AIDS virus” or“human immunodeficiency virus” are used herein to refer to these HIVviruses, and HIV-related and -associated viruses, generically. Moreover,a RNA virus of the subfamily Lentivirus preferably is a Visna/maedivirus (e.g., such as infect sheep), a feline immunodeficiency virus(FIV), bovine lentivirusu, simian immunodeficiency virus (SIV), anequine infectious anemia virus (EIAV), or a caprinearthritis-encephalitis virus (CAEV).

An especially preferred vector according to the invention is anadenoviral vector (i.e., a viral vector of the family Adenoviridae,optimally of the genus Mastadenovirus). Desirably such a vector is anAd2 or Ad5 vector, although other serotype adenoviral vectors can beemployed. The adenoviral vector employed for gene transfer can bewild-type (i.e., replication competent). Alternately, the adenoviralvector can comprise genetic material with at least one modificationtherein, which can render the virus replication deficient. Themodification to the adenoviral genome can include, but is not limitedto, addition of a DNA segment, rearrangement of a DNA segment, deletionof a DNA segment, replacement of a DNA segment, or introduction of a DNAlesion. A DNA segment can be as small as one nucleotide and as large as36 kilobase pairs (i.e., the approximate size of the adenoviral genome)or, alternately, can equal the maximum amount which can be packaged intoan adenoviral virion (i.e., about 38 kb). Preferred modifications to theadenoviral genome include modifications in the E1, E2, E3 or E4 region.Similarly, an adenoviral vector can be a cointegrate, i.e., a ligationof adenoviral sequences, with other sequences, such as other virus orplasmid sequences.

In terms of a viral vector (e.g., particularly a replication deficientadenoviral vector), such a vector can comprise either complete capsids(i.e., including a viral genome such as an adenoviral genome) or emptycapsids (i.e., in which a viral genome is lacking, or is degraded, e.g.,by physical or chemical means). Along the same lines, since methods areavailable for transferring viruses, plasmids, and phages in the form oftheir nucleic acid sequences (i.e., RNA or DNA), a vector (i.e., atransfer vector) similarly can comprise RNA or DNA, in the absence ofany associated protein such as capsid protein, and in the absence of anyenvelope lipid. Similarly, since liposomes effect cell entry by fusingwith cell membranes, a transfer vector can comprise liposomes (e.g.,such as are commercially available, for instance, from LifeTechnologies, Bethesda, Md.), with constitutive nucleic acids encodingthe coat protein. Thus, according to the invention whereas a vector“comprises” a chimeric adenoviral coat protein, a transfer vector“encodes” a chimeric adenoviral coat protein; liposome transfer vectorsin particular “encode” in the sense that they contain nucleic acidswhich, in fact, encode the protein.

A vector according to the invention can comprise additional sequencesand mutations, e.g., some within the coat protein itself. For instance,a vector according to the invention further preferably comprises anucleic acid comprising a passenger gene.

A “nucleic acid” is a polynucleotide (DNA or RNA). A “gene” is anynucleic acid sequence coding for a protein or a nascent RNA molecule. A“passenger gene” is any gene which is not typically present in and issubcloned into a vector (e.g., a transfer vector) according to thepresent invention, and which upon introduction into a host cell isaccompanied by a discernible change in the intracellular environment(e.g., by an increased level of deoxyribonucleic acid (DNA), ribonucleicacid (RNA), peptide or protein, or by an altered rate of production ordegradation thereof). A “gene product” is either an as yet untranslatedRNA molecule transcribed from a given gene or coding sequence (e.g.,mRNA or antisense RNA) or the polypeptide chain (i.e., protein orpeptide) translated from the mRNA molecule transcribed from the givengene or coding sequence. Whereas a gene comprises coding sequences plusany non-coding sequences, a “coding sequence” does not include anynon-coding (e.g., regulatory) DNA. A gene or coding sequence is“recombinant” if the sequence of bases along the molecule has beenaltered from the sequence in which the gene or coding sequence istypically found in nature, or if the sequence of bases is not typicallyfound in nature. According to this invention, a gene or coding sequencecan be wholly or partially synthetically made, can comprise genomic orcomplementary DNA (cDNA) sequences, and can be provided in the form ofeither DNA or RNA.

Non-coding sequences or regulatory sequences include promoter sequences.A “promoter” is a DNA sequence that directs the binding of RNApolymerase and thereby promotes RNA synthesis. “Enhancers” arecis-acting elements of DNA that stimulate or inhibit transcription ofadjacent genes. An enhancer that inhibits transcription is also termed a“silencer”. Enhancers differ from DNA-binding sites forsequence-specific DNA binding proteins found only in the promoter (whichare also termed “promoter elements”) in that enhancers can function ineither orientation, and over distances of up to several kilobase pairs,even from a position downstream of a transcribed region. According tothe invention, a coding sequence is “operably linked” to a promoter(e.g., when both the coding sequence and the promoter constitute apassenger gene) when the promoter is capable of directing transcriptionof that coding sequence.

Accordingly, a “passenger gene” can be any gene, and desirably is eithera therapeutic gene or a reporter gene. Preferably a passenger gene iscapable of being expressed in a cell in which the vector has beeninternalized. For instance, the passenger gene can comprise a reportergene, or a nucleic acid sequence which encodes a protein that can insome fashion be detected in a cell. The passenger gene also can comprisea therapeutic gene, for instance, a therapeutic gene which exerts itseffect at the level of RNA or protein. For instance, a protein encodedby a transferred therapeutic gene can be employed in the treatment of aninherited disease, such as, e.g., the cystic fibrosis transmembraneconductance regulator cDNA for the treatment of cystic fibrosis. Theprotein encoded by the therapeutic gene may exert its therapeutic effectby resulting in cell killing. For instance, expression of the gene initself may lead to cell killing, as with expression of the diphtheriatoxin A gene, or the expression of the gene may render cells selectivelysensitive to the killing action of certain drugs, e.g., expression ofthe HSV thymidine kinase gene renders cells sensitive to antiviralcompounds including acyclovir, gancyclovir and FIAU(1-(2-deoxy-2-fluoro-β-D-arabinofuranosil)-5-iodouracil).

Moreover, the therapeutic gene can exert its effect at the level of RNA,for instance, by encoding an antisense message or ribozyme, a proteinwhich affects splicing or 3′ processing (e.g., polyadenylation), or canencode a protein which acts by affecting the level of expression ofanother gene within the cell (i.e., where gene expression is broadlyconsidered to include all steps from initiation of transcription throughproduction of a processed protein), perhaps, among other things, bymediating an altered rate of mRNA accumulation, an alteration of mRNAtransport, and/or a change in post-transcriptional regulation.Accordingly, the use of the term “therapeutic gene” is intended toencompass these and any other embodiments of that which is more commonlyreferred to as gene therapy and is known to those of skill in the art.Similarly, the recombinant adenovirus can be used for gene therapy or tostudy the effects of expression of the gene in a given cell or tissue invitro or in vivo.

The recombinant adenovirus comprising a chimeric coat protein such as afiber protein and the recombinant adenovirus that additionally comprisesa passenger gene or genes capable of being expressed in a particularcell can be generated by use of a transfer vector, preferably a viral orplasmid transfer vector, in accordance with the present invention. Sucha transfer vector preferably comprises a chimeric adenoviral coatprotein gene sequence as previously described. The chimeric coat proteingene sequence comprises a nonnative sequence in place of the nativesequence, which has been deleted, or in addition to the native sequence.

A recombinant chimeric coat protein gene sequence (such as a fiber genesequence) can be moved from an adenoviral transfer vector intobaculovirus or a suitable prokaryotic or eukaryotic expression vectorfor expression and evaluation of receptor or protein specificity andavidity, trimerization potential, penton base binding, and otherbiochemical characteristics.

Accordingly, the present invention also provides recombinant baculoviraland prokaryotic and eukaryotic expression vectors comprising a chimericadenoviral coat protein gene sequence (preferably a fiber genesequence), which also are “transfer vectors” as defined herein. Thechimeric coat protein gene sequence (e.g., fiber gene sequence) includesa nonnative sequence in addition to or in place of a native amino acidsequence, and which enables the resultant chimeric coat protein (e.g.,fiber protein) to bind to a binding site other than a binding site boundby the native sequence. By moving the chimeric gene from an adenoviralvector to baculovirus or a prokaryotic or eukaryotic expression vector,high protein expression is achievable (approximately 5-50% of the totalprotein being the chimeric fiber).

A vector according to the invention further can comprise, either within,in place of, or outside of the coding sequence of a coat proteinadditional sequences that impact upon the ability of a coat protein suchas fiber protein to trimerize, or comprise a protease recognitionsequence. A sequence that impacts upon the ability to trimerize is oneor more sequences that enable trimerization of a chimeric coat proteinthat is a fiber protein. A sequence that comprises a proteaserecognition sequence is a sequence that can be cleaved by a protease,thereby effecting removal of the chimeric coat protein (or a portionthereof) and attachment of the recombinant adenovirus to a cell by meansof another coat protein. When employed with a coat protein that is afiber protein, the protease recognition site preferably does not affectfiber trimerization or receptor specificity of the fiber protein. Forinstance, in one embodiment of the present invention, preferably thefiber protein, or a portion thereof, is deleted in by means of aprotease recognition sequence, and then the novel cell surface bindingsite is incorporated into either the penton base or hexon coat protein,preferably with use of a spacer sequence as previously described.

In terms of the production of vectors and transfer vectors according tothe invention, transfer vectors are constructed using standard molecularand genetic techniques such as are known to those skilled in the art.Vectors (e.g., virions or virus particles) are produced using viralvectors. For instance, a viral vector comprising a chimeric coat proteinaccording to the invention can be constructed by providing to a cellthat does not comprise any E4 complementing sequences: (1) a linearvector comprising the chimeric fiber and the wild-type E4 gene, and (2)a linear vector that is E4⁻, as illustrated in FIG. 3. As described inthe Examples which follow, this methodology results in recombinationbetween the sequences, generating a vector that comprises a portion ofthe initial E4⁻ vector and a portion of the E4⁺ vector, particularly theregion comprising the chimeric fiber sequences.

Similarly, the fiber chimera-containing particles are produced instandard cell lines, e.g., those currently used for adenoviral vectors.Following production and purification, the particles in which fiber isto be deleted are rendered fiberless through digestion of the particleswith a sequence-specific protease, which cleaves the fiber proteins andreleases them from the viral particles to generate fiberless particles.For example, thrombin recognizes and cleaves at known amino acidsequences that can be incorporated into the vector (Stenflo et al., J.Biol. Chem., 257, 12280-12290 (1982)). Similarly, deletion mutantslacking the fiber gene can be employed in vector construction, e.g.,H2dl802, H2dl807, and H2dl1021 (Falgout et al., J. Virol., 62, 622-625(1988). These fiberless particles have been shown to be stable andcapable of binding and infecting cells (Falgout et al., supra). Theseresultant particles then can be targeted to specific tissues via thepenton base or other coat protein, preferably such other coat proteinthat comprises one or more nonnative amino acid sequences according tothe invention.

Alternately, recombinant adenovirus comprising chimeric fiber proteinhaving further modifications can be produced by the removal of thenative knob region, which comprises receptor-binding and trimerizationdomains, of the fiber protein and its replacement with a nonnativetrimerization domain (Peteranderl et al., Biochemistry, 31, 12272-12276(1992)) and a nonnative amino acid sequence according to the invention.A recombinant adenovirus comprising a chimeric fiber protein also can beproduced by point mutation in the knob region and the isolation ofclones that are capable of trimerization. In either case, and also withrespect to the removal and replacement of the native receptor-specificbinding sequence as described above, new protein binding domains can beadded onto the C-terminus of the fiber protein or into exposed loops ofthe fiber protein by inserting one or more copies of the nucleic acidsequence encoding the nonnative amino acid sequence into the appropriateposition. Preferably, such a fiber protein is able to trimerize, so thatit is able to bind to penton base protein.

The method described above for generating chimeric fiber protein alsocan be used to make other chimeric coat proteins, e.g., chimeric hexonor penton protein. For example, the RGD amino acid sequence of thepenton base protein is replaced at the DNA level with an amino acidbinding sequence for a given receptor, receptor-specific antibody domainor epitope. Alternatively, the RGD amino acid sequence is renderedinactive at the DNA level by mutation of the RGD amino acid sequence,such as by insertional mutagenesis, for example, or renderedconformationally inaccessible in the penton base protein, such as byinsertion of a DNA sequence into or adjacent to the adenoviral pentonbase gene sequence, wherein “gene sequence” refers to the completepenton base gene sequence as well as any lesser gene sequences that arecapable of being expressed as functional penton base protein.Preferably, the DNA sequence is inserted near the gene sequence encodingthe RGD amino acid sequence, so as to move the gene sequence encodingthe RGD amino acid sequence within the penton base gene sequence suchthat in the chimeric penton base protein the RGD amino acid sequence isconformationally inaccessible for binding to a receptor. In the lattercase, the inserted nonpenton base gene sequence that causes theconformational inaccessibility of the RGD amino acid sequence in thepenton base protein is preferably one that encodes an amino acidsequence that is specific for binding to a receptor, a receptor-specificantibody domain or epitope. Removal, inactivation, or the rendering ofthe RGD amino acid sequence conformationally inaccessible alters oreliminates the ability of the penton base molecule to bind anα_(v)integrin receptor.

Illustrative Uses

The present invention provides a chimeric protein that is able to bindto cells and mediate entry into cells with high efficiency, as well asvectors and transfer vectors comprising same. The chimeric coat proteinitself has multiple uses, e.g., as a tool for studies in vitro ofadenovirus binding to cells (e.g., by Scatchard analysis as shownpreviously by Wickham et al. (1993), supra), to block binding ofadenovirus to receptors in vitro (e.g., by using antibodies, peptides,and enzymes, as described in the Examples), and to protect againstadenoviral infection in vivo by competing for binding to the bindingsite by which adenovirus effects cell entry.

A vector comprising a chimeric coat protein also can be used in straingeneration and as a means of making new vectors. For instance, thenonnative amino acid sequence can bind to nucleic acids, and can beintroduced intracellularly as a means of generating new vectors viarecombination. Similarly, a vector can be used in gene therapy. Forinstance, a vector of the present invention can be used to treat any oneof a number of diseases by delivering to targeted cells corrective DNA,i.e., DNA encoding a function that is either absent or impaired, or adiscrete killing agent, e.g., DNA encoding a cytotoxin that, forexample, is active only intracellularly. Diseases that are candidatesfor such treatment include, for example, cancer, e.g., melanoma, gliomaor lung cancers; genetic disorders, e.g., cystic fibrosis, hemophilia ormuscular dystrophy; pathogenic infections, e.g., human immunodeficiencyvirus, tuberculosis or hepatitis; heart disease, e.g., preventingrestenosis following angioplasty or promoting angiogenesis to reperfusenecrotic tissue; and autoimmune disorders, e.g., Crohn's disease,colitis or rheumatoid arthritis.

In particular, gene therapy can be carried out in the treatment ofdiseases, disorders, or conditions associated with different tissuesthat ostensibly lack high levels of the receptor to which wild-typeadenovirus fiber protein binds, and thus for which currentadenoviral-mediated approaches to gene therapy are less than optimal(e.g., for delivery to monocyte/macrophages, fibroblasts, neuronal,smooth muscle, and epithelial cells). Tissues comprised of these cells(and diseases, disorders, or conditions associated therewith) include,but are not limited to: endothelia (e.g., angiogenesis, restenosis,inflammation, and tumors); neuronal tissue (e.g., tumors and Alzheimer'sdisease); epithelium (e.g., disorders of the skin, cornea, intestine,and lung); hematopoietic cells (e.g., human immunodeficiency virus(HIV-1, HIV-2), cancers, and anemias); smooth muscle (e.g., restenosis);and fibroblasts (e.g., inflammation).

Moreover, instead of transferring a therapeutic gene, a reporter gene,or some type of marker gene can be transferred instead using the vectors(particularly the adenoviral vectors) of the invention. Marker genes andreporter genes are of use, for instance, in cell differentiation andcell fate studies, as well as potentially for diagnostic purposes.Moreover, a standard reporter gene such as a β-galactosidase reportergene, a gene encoding green fluorescent protein (GFP), or aβ-glucuronidase gene can be used in vivo, e.g., as a means of assay in aliving host, or, for instance, as a means of targeted cell ablation(see, e.g., Minden et al., BioTechniques, 20, 122-129 (1996); Youvan,Science, 268, 264 (1995); U.S. Pat. No. 5,432,081; Deonarain et al., Br.J. Cancer, 70, 786-794 (1994)).

Similarly, it may be desirable to transfer a gene to use a hostessentially as a means of production in vivo of a particular protein.Along these lines, transgenic animals have been employed, for instancefor the production of recombinant polypeptides in the milk of transgenicbovine species (e.g., PCT International Application WO 93/25567). Other“non-therapeutic” reasons for gene transfer include the study of humandiseases using an animal model (e.g., use of transgenic mice and othertransgenic animals including p53 tumor suppressor gene knockouts fortumorigenic studies, use of a transgenic model for impaired glucosetolerance and human Alzheimer's amyloid precursor protein models for thestudy of glucose metabolism and pathogenesis of Alzheimer's disease,respectively, etc.)

These aforementioned illustrative uses are by no means comprehensive,and it is intended that the present invention encompasses such furtheruses which flow from, but are not explicitly recited, in the disclosureherein. Similarly, there are numerous advantages associated with the useof the various aspects of the present invention.

For instance, use of a universal targeting vector according to theinvention is advantageous inasmuch as: (1) the vector can potentially beused for all cells and tissues; (2) only one vector is required for usein all cell lines, there is no need for co-transfecting an independentvector; (3) the vector is capable of effecting gene delivery with anefficiency that is increased over that observed for vectors comprisingwild-type fiber protein; (4) the vector, unlike prior vectors, does nottarget specific cells, but instead increases transduction efficiency inwhat appears to be a global fashion; (5) the vector is capable ofmediating gene transfer when employed at a reduced dose (i.e.,multiplicity of infection (MOI)) as compared with vector comprisingwild-type fiber protein, and thus likely reduces the dosage-relateddrawbacks that accompany currently available adenoviral vectors; and (6)the vector can be propagated and maintained using currently availablecell lines.

The ability of a universal targeting vector such as a universaltargeting adenovirus vector to potentially bind to and enter all or mosttissues has several advantages. These advantages include increased genedelivery efficiency to multiple tissues, the availability of a singlevector capable of delivering genes to all tissues, and simplifiedproduction of necessary components for gene delivery. Moreover, such auniversal targeting vector comprises a potential to deliver exogenousDNA into cells by “piggy backing” the DNA on the vector by means of aprotein/DNA interaction.

Further potential advantages of such a universal targeting vectorinclude a substantially increased efficiency of delivery (e.g.,increased by 10- to 100-fold) into cells expressing low levels of fiberreceptor to which wild-type fiber protein binds, as well as increasedefficiency into cells or tissues expressing fiber receptor to whichwild-type fiber binds. Moreover the reduced dosage at which the vectorsare employed should result in a decrease in adenovirus-associatedinflammation, the humoral response to adenovirus, and the cytotoxicT-lymphocyte response to adenovirus.

Furthermore, the vector is advantageous in that it can be isolated andpurified by conventional means. Since changes in the vector are made atthe genome level, there are no cumbersome and costly post-productionmodifications required, as are associated with other vectors (see, e.g.,Cotten et al., supra; Wagner et al., supra). Similarly, specialreceptor-expressing cells lines are not required. A UTV vector can bepropagated to similar titers as a wild-type vector lacking the fibermodification.

Means of Administration

The vectors and transfer vectors of the present invention can beemployed to contact cells either in vitro or in vivo. According to theinvention “contacting” comprises any means by which a vector isintroduced intracellularly; the method is not dependent on anyparticular means of introduction and is not to be so construed. Means ofintroduction are well known to those skilled in the art, and also areexemplified herein.

Accordingly, introduction can be effected, for instance, either in vitro(e.g., in an ex vivo type method of gene therapy or in tissue culturestudies) or in vivo by electroporation, transformation, transduction,conjugation or triparental mating, (co-)transfection, (co-)infection,membrane fusion with cationic lipids, high velocity bombardment withDNA-coated microprojectiles, incubation with calcium phosphate-DNAprecipitate, direct microinjection into single cells, and the like.Similarly, the vectors can be introduced by means of cationic lipids,e.g., liposomes. Such liposomes are commercially available (e.g.,Lipofectin®, Lipofectamine™, and the like, supplied by LifeTechnologies, Gibco BRL, Gaithersburg, Md.). Moreover, liposomes havingincreased transfer capacity and/or reduced toxicity in vivo (see, e.g.,PCT patent application WO 95/21259) can be employed in the presentinvention. Other methods also are available and are known to thoseskilled in the art.

According to the invention, a “host” (and thus a “cell” from a host)encompasses any host into which a vector of the invention can beintroduced, and thus encompasses an animal, including, but not limitedto, an amphibian, bird, fish, insect, reptile, or mammal. Optimally ahost is a mammal, for instance, rodent, primate (such as chimpanzee,monkey, ape, gorilla, orangutan, or gibbon), feline, canine, ungulate(such as ruminant or swine), as well as, in particular, human.

Inasmuch as a universal targeting vector ostensibly enters all cells, acell can be any cell into which such a vector can enter. In particular,a universal targeting vector can be employed for gene transfer to a cellthat expresses low or undetectable levels of fiber receptor, including,but not limited to, an endothelial, smooth muscle, neuronal,hematopoietic, or fibroblast cell.

One skilled in the art will appreciate that suitable methods ofadministering a vector (particularly an adenoviral vector) of thepresent invention to an animal for purposes of gene therapy (see, forexample, Rosenfeld et al., Science, 252, 431-434 (1991); Jaffe et al.,Clin. Res., 39(2), 302A (1991); Rosenfeld et al., Clin. Res., 39(2), 311A (1991); Berkner, BioTechniques, 6, 616-629 (1988)), chemotherapy, andvaccination are available, and, although more than one route can be usedfor administration, a particular route can provide a more immediate andmore effective reaction than another route. Pharmaceutically acceptableexcipients also are well-known to those who are skilled in the art, andare readily available. The choice of excipient will be determined inpart by the particular method used to administer the recombinant vector.Accordingly, there is a wide variety of suitable formulations for use inthe context of the present invention. The following methods andexcipients are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

A vector or transfer vector of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Additionally, a vector or transfer vector of the present invention canbe made into suppositories by mixing with a variety of bases such asemulsifying bases or water-soluble bases.

Formulations suitable for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulascontaining, in addition to the active ingredient, such carriers as areknown in the art to be appropriate.

The dose administered to an animal, particularly a human, in the contextof the present invention will vary with the gene of interest, thecomposition employed, the method of administration, and the particularsite and organism being treated. However, the dose should be sufficientto effect a therapeutic response.

As previously indicated, a vector or a transfer vector of the presentinvention also has utility in vitro. Such a vector can be used as aresearch tool in the study of adenoviral attachment and infection ofcells and in a method of assaying binding site-ligand interaction.Similarly, the recombinant coat protein comprising a nonnative aminoacid sequence in addition to or in place of a native receptor bindingsequence can be used in receptor-ligand assays and as adhesion proteinsin vitro or in vivo, for example.

The following examples further illustrate the present invention and, ofcourse, should not be construed as in any way limiting its scope.

EXAMPLE 1

This example describes an investigation of the levels of adenovirusreceptor in different cells, as determined by the ability of wild-typeadenovirus to bind to the cells.

For these experiments, the ability of adenovirus comprising wild-typefiber to bind to cells derived from various tissues was assessed.Adenovirus particles of an Ad5 strain were labeled with [³H]-thymidineas previously described (see, e.g., Wickham et al., Cell, 73, 309-319(1993)). Subsaturating levels of thymidine-labeled adenovirus were addedto 200 μl of 10⁶ cells preincubated about 30 to 60 minutes with orwithout 20 μg/ml of soluble fiber protein. The cells were incubated withthe virus for 1 hour at 4° C. and then washed 3 times with coldphosphate buffered saline (PBS). The remaining cell-associated countswere measured in a scintillation counter. Specific binding was measuredby subtracting the cell-associated counts (i.e., counts per minute(cpm)) in the presence of fiber from the cell-associated counts in theabsence of fiber. Binding in the presence of fiber was never more than2% of the total input of radioactive virus particles. Results wereobtained as the average of triplicate measurements.

As illustrated in FIG. 1, a substantial number of the cells derived fromdifferent tissues expressed little or no fiber receptor, as indicated bya relative inability of wild-type adenovirus to bound to these cells.Cells of epithelial origin (i.e., “receptor-plus” cells including Chang,HeLa, and A549 cells) bound high levels of adenovirus. In comparison,non-epithelial cells (i.e., “receptor-minus” cells such asmonocyte/macrophages, fibroblasts, neuronal, smooth muscle, andepithelial cells) exhibited about 10-fold or more reductions in virusbinding as compared to epithelial-like cells.

These results confirm the previously unrecognized relative inability ofadenovirus to bind to and hence enter receptor-minus non-epithelialcells, as compared with receptor-plus epithelial cells. Presumably thisinability is due to the low representation of receptors for wild-typeadenoviral fiber protein on these cells.

EXAMPLE 2

This example describes the construction of an adenoviral vectorcomprising a chimeric coat protein, particularly a chimeric adenoviralfiber protein.

To overcome the transduction limitation imposed by the presence of onlya limited number of fiber receptors on clinically relevant tissues suchas non-epithelial tissue, a modified adenovirus vector was constructedas depicted in FIGS. 2A and 2B to derive a vector that is referred toherein as a “universal transfer vector”, or UTV. In particular, aframeshift mutation was created in a gene encoding an adenoviral coatprotein, in this case, in the fiber gene. In wild-type adenovirus, theunmodified fiber gene contains a nested translational stop signal (TAA)and transcriptional polyadenylation signal (AATAAA). The polyadenylationsignal directs the addition of a polyA tail onto the 3′ end of thetranscript. The polyA tail typically comprises anywhere from about 20 toabout 200 nucleotides. Following transcription and exit from thenucleus, the TAA stop signal directs termination of translation by theribosome.

In comparison, the modified fiber gene of a UTV vector lacks an in-frametranslational “stop” signal. Following normal transcription and additionof the polyA extension onto the mRNA, in the absence of the stop codon,the ribosome continues translation of the transcript into the polyAregion. Inasmuch as the codon AAA codes for the amino acid lysine, theresultant chimeric fiber gene translation product produced by a UTVcontains an addition of a string of polylysine residues at theC-terminus, i.e., Lys Lys Lys Lys Lys Lys Lys Lys [SEQ ID NO:1]. It ispossible that a cellular process acts to limit the length of thepolylysine string, since the polylysine residues typically comprise fromabout 3 to about 30 residues in the chimeric fiber protein. Whatever thecase, however, the polylysine protein modification, as well as furthermodifications described herein, allows the UTV to efficiently attach tocells lacking high levels of the receptor for wild-type adenoviral fiberprotein (i.e., receptor-minus cells).

In terms of vector construction and characterization, standard molecularand genetic techniques, such as the generation of strains, plasmids, andviruses, gel electrophoresis, DNA manipulations including plasmidisolation, DNA cloning and sequencing, Western blot assays, and thelike, were performed such as are known to those skilled in the art, andas are described in detail in standard laboratory manuals (e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (ColdSpring Harbor, N.Y., 1992); Ausubel et al., Current Protocols inMolecular Biology, (1987)). Restriction enzymes and other enzymes usedfor molecular manipulations were purchased from commercial sources(e.g., Boehringer Mannheim, Inc., Indianapolis, Ind.; New EnglandBiolabs, Beverly, Mass.; Bethesda Research Laboratories, Bethesda, Md.),and were used according to the recommendations of the manufacturer.Cells employed for experiments (e.g., cells of the transformed humanembryonic kidney cell line 293 (i.e., CRL 1573 cells) and other cellssupplied by American Type Culture Collection) were cultured andmaintained using standard sterile culture reagents, media andtechniques, as previously described (Erzerum et al., Nucleic AcidsResearch, 21, 1607-1612 (1993)).

Accordingly, the frameshift mutation of the fiber stop codon was createdby introducing a modified BamHI site (i.e., GGATCCAA [SEQ ID NO:6]) intoan adenoviral transfer vector. This was done as illustrated in FIG. 3 bystarting with the transfer plasmid pAd NS 83-100 (which also is known asp193NS 83-100 or pNS 83-100). pAd NS 83-100 was constructed by cloningthe Ad5 NdeI to SalI fragment, which spans the 83-100 map unit region ofthe Ad5 genome containing the fiber gene, into the plasmid pNEB 193 (NewEngland Biolabs, Beverly, Mass.).

The NdeI-MunI fragment of pAd NS 83-100 was replaced with a syntheticoligonucleotide comprising a BamHI site, which was flanked by a 5′ NdeIsite and a 3′ MunI site to facilitate cloning. The double-strandedsynthetic oligonucleotide fragment was created from overlappingsynthetic single-stranded sense and antisense oligonucleotides, i.e.,respectively, the sense primer TAT GGA GGA TCC AAT AAA GAA TCG TTT GTGTTA TGT TTC AAC GTG TTT ATT TTT C [SEQ ID NO:9], and the antisenseprimer AAT TGA AAA ATA AAC ACG TTG AAA CAT AAC ACA AAC GAT TCT TTA TTGGAT CCT CCA [SEQ ID NO:10], as illustrated in FIGS. 4A and 4B,respectively. The ends of the overlapping oligomers were made to haveoverhangs compatible for direct cloning into the NdeI and MunI sites.

The resultant transfer plasmid, pAd NS 83-100 (F) (which also is knownas p193NS (ΔF) or pNS (ΔF)), lacks all but the first 50 base pairs ofthe coding sequence for the fiber gene (i.e., is “fiber-minus”). Thevector furthermore contains the entire adenovirus E4 coding sequence.The plasmid retains the AATAAA polyadenylation signal included in thesynthetic NdeI/MunI oligonucleotide, and also incorporates the new BamHIrestriction site.

The mutated fiber gene was incorporated into the fiber-minus pAd NS83-100 plasmid using synthetic sense and antisense oligonucleotideprimers to amplify the fiber gene with use of the polymerase chainreaction (PCR) while incorporating a modified BamHI site following thelast codon of the fiber gene to create the mutant fiber gene. Thisincorporated modified BamHI site also serves to code for the amino acidsglycine and serine, resulting in a chimeric nucleic acid sequence of GGATCC AAT AAA GAA TCG TTT GTG TTA TGT [SEQ ID NO:7]. The modified fibergene thus codes for an extension to the resultant chimeric fiber proteinof Gly Ser Asn Lys Glu Ser Phe Val Leu Lys Lys Lys [SEQ ID NO:4],wherein the length of the polylysine string can vary. The syntheticoligonucleotides employed for fiber amplification were the primer TCCCCCCGGG TCTAGA TTA GGA TCC TTC TTG GGC AAT GTA TGA [SEQ ID NO:1], and theprimer CGT GTA TCC ATA TGA CAC AGA [SEQ ID NO:12], as illustrated inFIGS. 4C and 4D, respectively.

The amplified gene product was then cut with the restriction enzymesNdeI and BamHI, and was cloned into the NdeI/BamHI sites of thefiber-minus plasmid pAd NS 83-100 to create the transfer vector pAd NS83-100 UTV (which also is known as p193NS (F5*), p193 (F5*), or pNS(F5*)). The entire NdeI to SalI adenovirus sequence of pAd NS 83-100 UTVwas cloned into the fiber-minus plasmid pAd BS 59-100 to create pAd BS59-100 UTV (which also is known as p193NS (F5*), p193 (F5*), or pNS(F5*).

The UTV adenovirus vector was created through homologous recombinationin 293 cells. Namely, the E4⁺ pAd BS 59-100 UTV transfer vector waslinearized with SalI, and was transfected into 293 cells that werepreviously infected with the adenovirus vector, A2F. The A2F vector wasderived from a GV10 vector. The Ad5-based vector GV10 contains the lacZgene under the control of the Rous sarcoma virus promoter (i.e.,comprises RSV lacZ). The insertion of the reporter gene in GV10 is madewithin the E1 region (i.e., the vector is E1⁻). The GV10 vector alsocontains a deletion of the E3 region, but is E4⁺. In comparison withGV10 (i.e., RSV lacZ E1⁻ E3⁻ E4⁺), A2F further comprises a deletion ofthe essential E4 adenovirus genes, but is E3⁺ (i.e., RSV lacZ E1⁻ E3⁺E4¹ ).

The 293 cells contain an E1 complementing sequence, but do not containan E4 complementing sequence. The lack of an E4 complementing sequenceprevents replication of the E4⁻ A2F vector in the 293 cell line.However, upon co-introduction of A2F virus and pAd BS 59-100 UTV in 293cells, homologous recombination takes place between the UTV transfervector and the A2F adenoviral genome, producing an E3⁺ E4⁺ adenovirusgenome comprising a chimeric fiber protein, which is capable ofreplication in 293 cells. This particular resultant UTV vector wasdesignated GV10 UTV.

The GV10 UTV vector was isolated using standard plaque isolationtechniques with 293 cells. Following three successive rounds ofplaque-purification, the GV10 UTV vector contained the fiber mutationand was free of any contamination by the E4⁻ A2F vector. The presence ofthe chimeric fiber sequences in the GV10 UTV vector was confirmed bysequencing the fiber mRNA using reverse transcriptase-polymerase chainreaction (RT-PCR), which validated the presence of a polyadenine tail inthe chimeric fiber mRNA.

Similarly, the production of a chimeric fiber protein by the vector wasconfirmed by Western blot. To accomplish this, 293 cells were infectedat a multiplicity of infection (MOI) of 5 with either GV10 comprisingwild-type adenoviral fiber protein or with GV10 UTV comprising chimericfiber protein. At two days post-infection, the cells were washed andthen lysed in PBS by three freeze-thaw cycles. The lysates were clearedby centrifugation and loaded onto a 10% sodium dodecylsulfate/polyacrylamide gel. Following electrophoresis, the proteins weretransferred onto nitrocellulose and detected by chemiluminescence usinga polyclonal antibody to fiber. The Western blot is depicted in FIG. 5.As can be seen from this figure, the migration of the proteins indicatesthat the chimeric UTV fiber is about 1.5 to about 2.0 kilodaltons largerthan the unmodified 62 kilodalton WT fiber protein.

These results confirm that the method identified herein can be employedto introduce modifications into the fiber protein to produce a chimericfiber protein. Similar techniques can be employed to introducemodifications into the hexon or penton proteins, or to introduce similarmodifications (e.g., the addition of a string of amino acids comprisedof arginine, lysine and/or histidine, or comprised of aspartate and/orglutamate, or the addition of any of these sequences into a codingregion of the coat proteins).

EXAMPLE 3

This example describes the binding to cells of an adenoviral vectorcomprising a chimeric coat protein such as a chimeric fiber protein ascompared with a wild-type adenoviral vector, either in the presence orabsence of added soluble wild-type fiber protein

For these experiments, the cells identified in Example 1 to whichadenovirus binds with either high efficiency (i.e. receptor-plus cells)or low efficiency (i.e., receptor-minus cells) were employed. Theepithelial cell line A549 was used as representative of receptor-pluscells, and the fibroblast cell line HS 68 was used as representative ofreceptor-minus cells. Confluent monolayers of either A549 or HS 68 cellswere preincubated at 4° C. with concentrations of soluble fiber proteinranging from 0 to about 10 μg/ml. The GV10 UTV vector comprisingchimeric fiber protein (UTV) or GV10 vector comprising wild-type fiberprotein (WT) were labeled with tritiated thymidine as described inExample 1. About 20,000 cpm of [³H]-thymidine labeled GV10 UTV or GV10vector were then incubated with the cells for about 2 hours at 4° C. Thecells were washed three times with cold PBS, and the cell-associated cpmwere determined by scintillation counting. Results were obtained as theaverage of duplicate measurements and are presented in FIGS. 6A and 6Bfor the A549 and HS 68 cell lines, respectively.

As can be seen in FIGS. 6A-B, the GV10 UTV vector chimeric fiber proteinwas able to bind both receptor-plus (FIG. 6A) and receptor-minus (FIG.6B) cells with high efficiency. In comparison, the GV10 vectorcomprising wild-type fiber was more effective at binding toreceptor-plus cells. In particular, radiolabeled GV10 UTV bound to cellsexpressing detectable levels of fiber receptor (i.e., A549 alveolarepithelial cells) about 2- to 2.5-fold better than GV10. Whereas all ofthe binding of the GV10 vector was inhibited by competing recombinantfiber protein, only about 40% of the GV10 UTV vector was inhibited bythe addition of competing fiber. No detectable binding of GV10 vectorcomprising wild-type adenoviral fiber to HS 68 human foreskin fibroblastcells lacking fiber receptor was observed. In comparison, the GV10 UTVvector efficiently bound to HS 68 cells, and the addition of competingfiber protein had no effect on binding.

These results confirm that binding of the GV10 UTV vector comprising achimeric coat protein (i.e., a chimeric fiber protein) does not occurvia the wild-type adenoviral fiber receptor, and instead occurs via aheretofore unrecognized fiber receptor. Moreover, the results confirmthat incorporation of a chimeric coat protein such as a chimeric fiberprotein into an adenoviral vector results in an improved adenoviralvector. Namely, the modification comprised by the GV10 UTV vectorenables it overcome the aforementioned relative inability of wild-typeadenovirus to bind to receptor-minus cells, in particular,non-epithelial cells, and also allows the modified vector to bind toreceptor-plus cells with an increased efficiency.

EXAMPLE 4

This example describes an investigation of the ability of varioussoluble factors, and inhibitors of these soluble factors, to blockbinding of adenovirus comprising chimeric fiber protein toreceptor-minus HS 68 fibroblast cells.

For these experiments, the inhibition of GV10 UTV binding by variousnegatively charged molecules including salmon sperm DNA, mucin,chondroitin sulfate, and heparin, was assessed. Chondroitin sulfate andheparin are negatively charged molecules which get their charge fromsulfate groups. Mucin is negatively charged due to the presence ofsialic acid moieties, and DNA is negatively charged due to itsincorporation of phosphate moieties. About 20,000 cpm of UTV in 250 μlof binding buffer (i.e., Dulbecco's Modified Eagle Media (D-MEM) wasincubated at room temperature for about 30 minutes with concentrationsof negatively charged molecules ranging from about 1×10⁻³ to about 1×10⁴μg/ml. Following incubation, the mixtures were chilled on ice, and werethen added to prechilled HS 68 cells plated in 24 well plates. The cellswere incubated for about 1 hour, and then the cells were washed threetimes with PBS. Cell-associated cpm were determined by scintillationcounting, and reported as the average of duplicate measurements.

As indicated in FIG. 7, whereas the presence of competing wild-typefiber protein had no effect on binding of a GV10 UTV vector (i.e.,comprising chimeric fiber) to HS 68 cells, negatively-charged competingmolecules were able to block GV10 UTV binding. All four molecules wereable to inhibit GV10 UTV binding to HS 68 cells, although heparin andDNA were most effective. These molecules have no significant effect onthe binding of a GV10 vector (i.e., comprising wild-type fiber) to cellsexpressing high levels of fiber receptor (i.e., A549 cells; data notshown).

These results confirm that negatively charged molecules are able toblock binding of the GV10 UTV vector to cells mediated by chimeric fiberprotein. This inhibition presumably is due to the binding of thenegatively charged molecules to the positively charged polylysineresidues present on the GV10 UTV fiber. Accordingly, the impact ofenzymes which cleave these negatively charged molecules on binding tocells of the GV10 UTV vector was assessed.

HS 68 cells were plated in 24 well plates, and were preincubated withthe dilutions of heparinase (Sigma, St. Louis, Mo.), chondroitinase(Sigma), and sialidase (Boehringer Mannheim, Inc.) ranging from about0.0001 to 1 for 45 minutes at 37° C., followed by 15 minutes at 4° C.Whereas chondroitinase cleaves chondroitin sulfate, heparinase cleavesheparin and heparin sulfate, and sialidase cleaves sialic acid. Theinitial starting concentrations for dilutions were as follows:heparinase, 25 U/ml (U=0.1 μmole/hour, pH=7.5, 25° C.); chondroitinase,2.5 U/ml (U=1.0 μmole/minute, pH=8.0, 37° C.); and sialidase 0.25 U/ml(U=1.0 μmole/minute, pH=5.5, 37° C.). Following incubation, the cellswere washed three times with cold PBS, and were then incubated with20,000 cpm of labeled GV10 UTV vector for about 1 hour at 4° C. Thecells were then washed three times with cold PBS, and thecell-associated cpm were determined by scintillation counting. Theresults were reported as the average of duplicate measurements.

As illustrated in FIG. 8, pretreatment of HS 68 cells with enzymes thatremove negatively charged molecules from the cell surface confirms thatthe GV10 UTV vector comprising the chimeric fiber protein interacts withnegatively charged sites on the cell surface. In particular, heparinaseand sialidase were both able to reduce GV10 UTV binding, althoughheparinase was more effective than sialidase on HS 68 cells.

Thus, these results confirm that a vector comprising chimeric fiberprotein (e.g. a GV10 UTV vector), unlike wild-type adenovirus, interactsin a novel fashion with negatively charged molecules on the cell surfaceto effect cell entry. These results further demonstrate that a vectorcomprising negatively charged residues (e.g., aspartate and glutamate)instead of positively charged molecules (e.g., lysine) similarly can beemployed to bind to and effect cell entry via positively chargedmolecules present on the cell surface.

EXAMPLE 5

This example evaluate gene delivery to different types of cells mediatedby an adenoviral vector comprising chimeric coat protein such aschimeric fiber protein (e.g., GV10 UTV) as compared to gene deliverymediated by adenovirus comprising wild-type coat protein such as fiberprotein (e.g., GV10).

For these experiments, the relative levels of lacZ gene delivery by avector containing the wild-type fiber protein (i.e., GV10) as comparedwith vector containing chimeric fiber protein (i.e., GV10 UTV) werecompared in epithelial-like cells (i.e., HeLa, A549, HepG2 and H700 Tcells), smooth muscle cells (i.e., HA SMC and HI SMC cells), endothelialcells (i.e., HUVEC and CPAE cells), fibroblast cells (i.e., HS 68 andMRC-5 cells), glioblastoma cells (i.e., U118 cells) and monocytemacrophages (i.e., THP-1 cells). Approximately 2×10⁵ cells wereinoculated one day prior to transduction by adenovirus into 24 multiwellplates. Each well was then infected at an MOI of 1 with GV10 (i.e.,comprising wild-type adenoviral fiber protein) or with GV10 UTV (i.e.,comprising chimeric adenoviral fiber) in a 250 μl volume for about onehour. The wells were then washed and incubated for two days, after whichthe lacZ activity of the cell lysates was determined. The results werereported as the average of duplicate measurements.

As illustrated in FIG. 9, the use of the GV10 UTV vector to transfer areporter gene to a panel of cell lines confirms that the presence of thechimeric fiber protein (UTV) increases lacZ gene delivery to cellsexpressing low or undetectable levels of fiber receptor (i.e.,receptor-minus cells) from about 5- to about 300-fold as compared withwild-type vector (GV10). In cells expressing high levels of the fiberreceptor (i.e., receptor-plus cells), the incorporation of the chimericfiber protein in the GV10 UTV vector results in an increase in genedelivery of up to about 3-fold.

This reduction in expression observed with transduction ofreceptor-minus non-epithelial cells as compared with receptor-plusepithelial cells by adenovirus comprising a wild-type fiber protein(i.e., GV10) directly correlates with the relative ability of the vectorto bind these different cell types, as reported in Example 3. Theseresults support the view that the low expression of receptors forwild-type adenovirus fiber protein is a significant limiting factor totheir efficient transduction by current adenovirus vectors.

Similarly, the ability of the chimeric coat protein (i.e., the chimericfiber protein) to augment gene transfer in vivo was assessed. ThreeBALB/c mice were inoculated intranasally with about 1×10⁸ pfu ofGV10 in50 μl of a saline solution comprising 10 mM MgCl₂ and 10 mM Tris (pH7.8). Another three mice received the same dose of GV10 UTV, and twomice received the saline solution alone. The animals were sacrificed attwo days post-administration, and the lungs were assayed for lacZactivity. The lungs were prepared for analysis by snap-freezing the lungin liquid nitrogen, grinding the tissue with a mortar and pestle, andlysing the ground tissue in 1.0 ml of lacZ reporter lysis buffer(Promega Corp., Madison, Wis.). A fluorometric assay was used to monitorlacZ activity, and the results of the experiments were reported as theaverage activity measured from each group of animals.

The results of these experiments are illustrated in FIG. 10. As can beseen from this Figure, gene transfer in vivo mediated by the GV10 UTVvector comprising chimeric fiber protein (UTV) as compared with a vectorcomprising wild-type fiber protein (GV10) resulted in an average of8-fold higher delivery to mouse lung.

These results thus confirm that incorporation of a chimeric coat protein(in this case, a chimeric fiber protein) in an adenoviral vectorsubstantially increases the efficiency of vector-mediated gene deliveryboth in vitro and in vivo as compared to an adenovirus vector comprisingwild-type fiber protein. Moreover, the results support the conclusionthat low fiber receptor expression is a significant factor contributingto the suboptimal delivery observed in the lung and in other tissues.Also, the results confirm the superiority of the GV10 UTV vector, aswell as other similar UTV vectors, over other currently availableadenoviral vectors for gene transfer (e.g., delivery of the CFTR gene)to the lung and other tissues.

EXAMPLE 6

This example evaluates the ability of a vector according to theinvention comprising a chimeric coat protein (e.g., a chimeric fiberprotein) to interact with passenger DNA by means of a protein/DNAinteraction, and to thereby carry the DNA into the cell in a“piggy-back” fashion.

For these experiments, an adenoviral vector comprising wild-type fiber(i.e., GV10) and an adenoviral vector comprising chimeric fiber (i.e.,GV10 UTV) were used to assess gene transfer to receptor-plus epithelialcells (i.e., 293, A549, and H700 T cells). In control experiments, thecells were transduced with the vectors as previously described. In theexperimental condition, the vectors were incubated with the plasmidpGUS, which comprises a β-glucuronidase reporter gene, such that thechimeric adenoviral fiber protein was able to complex with the plasmidDNA. Specifically, about 5×10⁷ active particles (i.e., fluorescencefocus units (ffu)) of GV10 or GV10 UTV were incubated for 1 hour withabout 2.5 μg of plasmid pGUS DNA. The mixture was then added to about2×10⁵ of the indicated cells in 250 μl of DMEM containing 10% fetalbovine serum. Both β-glucuronidase and β-galactosidase activity werethen assessed by fluorometric assay at 10 days post-transduction.β-glucuronidase expression in cells was monitored similarly to theβ-galactosidase assay for lacZ expression, by monitoring the generationof a blue color when β-glucuronidase catalyzes a reaction with thesubstrate X-glu.

The results of these experiments are illustrated in FIG. 11. Comparablelevels of lacZ expression were obtained when either a GV10 vector (i.e.comprising wild-type fiber protein) or a GV10 UTV vector (i.e.comprising chimeric fiber protein) were employed to transfer thereporter gene in cis to epithelial cells. In comparison, the wild-typevector was able to transfer intracellularly the plasmid pGUS at only arelatively low level in all epithelial cells, as assessed byβ-glucuronidase gene expression. This basal level of gene transferlikely was accomplished by means of receptor-mediated uptake (RME) ofbystander molecules, as previously described (PCT patent application WO95/21259). However, with use of a GV10 UTV vector comprising a chimericfiber protein, transfer of the pGUS plasmid was substantially increased.In the case of gene transfer to 293 cells, pGUS plasmid-directedP-glucuronidase expression exceeded expression observed following GV10UTV-vector mediated transfer of a cis-linked reporter gene.

These results confirm that a vector comprising a chimeric coat proteinsuch as a chimeric fiber protein according to the invention demonstratesincreased transfer of a nucleic acid that is not located in cis with thevector. Ostensibly, this enhanced gene transfer is effected by theoccurrence of a protein/DNA interaction between the negatively chargedresidues on the chimeric fiber (e.g., residues of the polylysinestring), resulting in binding to the vector of the nucleic acid;however, other means of enhancement also are possible.

EXAMPLE 7

This example describes the construction of further plasmids containingUTV or UTV-like sequences in the C-terminus of the fiber protein.

The transfer plasmid, p193(F5*) (FIG. 12; also known as p193NS (F5*),pNS (F5*), and pAd NS 83-100 UTV) described in Example 2 was employed asa starting point for the construction of these further plasmidscontaining chimeric adenovirus fiber proteins. As depicted in FIG. 12,p193 (F5*) contains a mutated fiber gene with a BamHI site between thelast fiber protein codon and the frameshifted fiber protein stop codon.The further mutant transfer plasmids constructed as described hereincontain sequences in the fiber C-terminus encoding an amino acidglycine/serine repeat linker, a targeting sequence, and a stop codon.These plasmids were made by cloning synthetic oligonucleotides into theBamHI site of p193(F5*) to create the transfer plasmid p193NS (F5*)pGS(K7)(also known as p193 (F5*) pGS(K7) or pNS (F5*) pK7) depicted inFIG. 13.

Thus, the sequence of the wild-type Ad5 fiber gene is:

TCA TAC ATT GCC CAA GAA TAA AAA AGAA [SEQ ID NO:59] Ser Tyr Ile Ala GlnGlu [SEQ ID NO:6O]wherein “TAA” is a termination codon, and the polyadenylation sequenceis emboldened. The C-terminus of the mutated fiber gene present inp193(F5*) is:

[SEQ ID NO:19] TCA TAC ATT GCC CAA GAA GGA TCC  AA TAAA GAA [SEQ IDNO:20] Ser Tyr Ile Ala Gln Glu Gly Serwherein the underlined sequence indicates the mutated BamHI siteintroduced into the fiber protein, and the polyadenylation sequence isemboldened. In comparison, the amino acid sequence of the C-terminus ofthe fiber gene present in p193NS (F5*) pGS(K7) is:

G S G S G S G S G S KKKKKKK [SEQ ID NO:22]wherein the underlined sequence indicates the mutated BamHI siteintroduced into the fiber protein, and the emboldened sequence indicatesthe polylysine string added to the C-terminus. This amino acid sequenceis encoded by the nucleic acid sequence: GGA TCA GGA TCA GGT TCA GGG AGTGGC TCT AAA AAG AAG AAA AAG AAG AAG TAA [SEQ ID NO:21], wherein “TAA” isa termination codon.

The overlapping synthetic oligonucleotides used to make the transferplasmid p193NS (F5*) pGS(K7) were: pK7s (sense), GA TCA GGA TCA GGT TCAGGG AGT GGC TCT AAA AAG AAG AAA AAG AAG AAA TAA G [SEQ ID NO:61]; pK7a(antisense), GA TCC TTA CTT CTT CTT TTT CTT CTT TTT AGA GCC ACT CCC TGAACC TGA TCC T [SEQ ID NO:62]. The sense and antisense oligonucleotideswere mixed in equimolar ratios and cloned into the BamHI site of p193NS(F5*) to create p193NS (F5*) pGS(pK7). Verification of thecorrectly-oriented insert in p193NS (F5*) pGS(pK7) was performed by PCRusing the pK7s sense primer and the downstream antisense oligonucleotideprimer A5a32938, CAGGTTGAATACTAGGGTTCT [SEQ ID NO:63]. The plasmid wasalso verified to contain the correctly oriented insert by sequencing theDNA sequence in the region of the insert using the A5a32938 primer.

The transfer plasmid p193NS (F5*) was employed in the construction offurther mutant transfer plasmids that additionally contain a UTV orUTV-like cell targeting sequence in the C-terminus of the fiber protein.These plasmids include p193NS (F5*) pGS(null) (also known as p193 (F5*)pGS(null) or p193 (F5*) pGS), pBSS 75-100 pGS (null), pBSS 75-100pGS(RK32), pBSS 75-100 pGS(RK33), and pBSS 75-100 pGS(tat).

To construct p193NS (F5*) pGS(null), the complementary overlappingoligonucleotides pGSs, GATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGTTAAA [SEQ IDNO:64], and pGSa, GATCTTTAACTAGTCGAGCCACTGCCAGATCCTGAACCG [SEQ ID NO:65]were constructed for direct ligation into the BamHI-digested p193NS(F5*) plasmid. Verification of the correctly-oriented clone wasperformed by PCR using the pGSs primer and the downstream antisenseoligonucleotide primer A5a32938. The plasmid was also verified tocontain the correctly oriented insert by sequencing the DNA sequence inthe region of the insert using the A5a32938 primer.

The vector pBSS 75-100 pGS(null) (also known as pBSS 75-100 ΔE3pGS(null)) depicted in FIG. 14 was constructed by replacing the NheI toSalI fragment from pBSS 75-100 with the corresponding fragment fromp193NS (F5*) pGS(null). The SpeI site that is not within the fiberchimera gene was then eliminated by partially restricting the plasmidwith SpeI, filling in with Klenow fragment and then religating thevector. The resultant vector comprises the relevant nucleic acidsequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGTTAA [SEQ IDNO:23] (wherein “TAA” is a termination codon), which codes for the aminoacid sequence Ala Gln Glu Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser ThrSer [SEQ ID NO:24].

The inserts of plasmids pBSS 75-100 pGS(RK32) (also known as pBSS 75-100ΔE3 pGS(RKKK)₂ or pBSS 75-100 ΔE3 pGS(RKKK2)), pBSS 75-100 pGS(RK33)(also known as pBSS 75-100 ΔE3 pGS(RKKK)₃ or pBSS 75-100 ΔE3pGS(RKKK3)), and pBSS 75-100 pGS(tat), were constructed for directligation into the SpeI-digested pBSS 75-100 pGS(null) plasmid. Toconstruct pBSS 75-100 pGS(RK32) depicted in FIG. 15, the complementaryoverlapping oligonucleotides, RK32s, CTAGAAAGAAGAAACGCAAAAAGAAGA [SEQ IDNO:66] and RK32a, CTAGTCTTCTTTTTGCGTTTCTTCTTT [SEQ ID NO:67] wereemployed. The resultant vector comprises the relevant nucleic acidsequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGAAAGAAGAAACGCAAAAAGAAGACTAGTTAA [SEQ ID NO:25] (wherein “TAA” is a terminationcodon), which codes for the amino acid sequence Ala Gln Glu Gly Ser GlySer Gly Ser Gly Ser Gly Ser Thr Arg Lys Lys Lys Arg Lys Lys Lys Thr Ser[SEQ ID NO:26].

To construct pBSS 75-100 pGS(RK33) depicted in FIG. 16, thecomplementary overlapping oligonucleotides, RK33s,CTAGAAAGAAGAAGCGCAAAAAAAAAGAAAGAAGAAGA [SEQ ID NO:68] and RK33a,CTAGTCTTCTTCTTTCTTTTTTTTTTGCGCTTCTTCTTCTTT [SEQ ID NO:69], wereemployed. The resultant vector comprises the relevant nucleic acidsequence: GCCCAAGAAGGATCCGGTTCAGGATCTGGCAGTGGCTCGACTAGAAAGAAGAAGCGCAAAAAAAAAGAAAGAAGAAGACTAGTTAA [SEQ ID NO:27] (wherein “TAA” is atermination codon), which codes for the amino acid sequence Ala Gln GluGly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Arg Lys Lys Lys Arg Lys LysLys Arg Lys Lys Lys Thr Ser [SEQ ID NO:28].

To construct pBSS 75-100 pGS(tat) the complementary overlappingoligonucleotides, TATs, CT AGT TAT GGG AGA AAA AAG CGC AGG CAA CGA AGACGG GCA T [SEQ ID NO:70] and TATa, CT AGA TGC CCG TCT TCG TTG CCT GCGCTT TTT TCT CCC ATA A [SEQ ID NO:71] were employed. The resultant vectorcomprises the relevant nucleic acid sequence: ACT AGT TAT GGG AGA AAAAAG CGC AGG CAA CGA AGA CGG GCA TCT AGT [SEQ ID NO:72], which codes forthe amino acid sequence Thr Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg ArgArg Ala Ser Ser [SEQ ID NO:73].

Verification of the correctly-oriented clone was performed by PCR usingthe sense primers (RK32s, RK33s, or TATs) for each of the threerespective plasmids, and using the downstream antisense oligonucleotideprimer A5a32938. Each of the plasmids also were verified to contain thecorrectly-oriented insert by sequencing the DNA sequence in the regionof the insert using the A5a32938 primer.

Example 8

This example described the construction of plasmids containing UTVdomains in the fiber loop.

Plasmids containing a UTV sequence (and/or a spacer sequence) in anexposed loop of the fiber protein are constructed by incorporating anyof the aforesaid sequences (as well as any further UTV-like sequences)into the fiber protein. This is accomplished making use of the plasmidtransfer vector p193NS (F5*) to construct the further transfer vectorp193NS F5F2K (also called p193 F5F2K) depicted in FIG. 17. Plasmidp193NS F5F2K contains a unique Spe I restriction site within the Ad2fiber gene encoding an exposed loop in the protein. Namely, the fibergene present in p193NS F5F2K comprises the fiber sequence:

ATT ACA CTT AAT GGC ACT AGT GAA TCC [SEQ ID NO:29] Ile Thr Leu Asn GlyThr Ser Glu Ser [SEQ ID NO:30] ACA GAA ACT Thr Glu Thrwherein the underlined sequence indicates the novel Spe I siteintroduced into the fiber gene.

This vector was then used to clone targeting sequences into the Spe Isite. In particular, a nucleic acid sequence encoding the stretch of 8basic amino acids RKKKRKKK (Arg Lys Lys Lys Arg Lys Lys Lys [SEQ IDNO:74]) comprising the heparin binding domain were cloned into the Spe Isite of p193 F5F2K using overlapping sense and antisenseoligonucleotides.

Namely, the (RKKK)₂ sequence comprises, in part, the sequence:

TCT AGA AAA AAA AAA CGC AAG AAG AAG [SEQ ID NO:75] Thr Arg Lys Lys LysArg Lys Lys Lys [SEQ ID NO:76] ACT AGT Thr Ser.

The 27-mer sense oligonucleotide RK32s and 27-mer antisenseoligonucleotide RK32a described in Example 7 were employed for cloningthe PolyGS(RKKK)₂ sequence comprising the RKKKRKKK [SEQ ID NO:74]peptide motif. The p193NS F5F2K(RKKK)₂ plasmid was constructed bycloning the DNA sequence encoding the binding domain into the Spe I siteof p193NS F5FK2. The overlapping sense and antisense oligonucleotidesencoding the binding domain were first annealed and then directlyligated into the Spe I restriction site to result in the plasmid p193NSF5F2K(RKKK)₂ depicted in FIG. 18. This plasmid also is known as p193NSF5F2K(RKKK2), p193NS F5F2K(RK32), or p193 F5F2K(RKKK2). The relevantportion of the modified loop of the fiber knob present in p193NSF5F2K(RKKK)₂ is:

ATT ACA CTT AAT GGC ACT AGA AAG AAG [SEQ ID NO:31] Ile Thr Leu Asn GlyThr Arg Lys Lys [SEQ ID NO:32] AAA CGC AAA AAG AAG ACT AGT GAA TCC LysArg Lys Lys Lys Thr Ser Glu Ser ACA GAA ACT Thr Glu Thr.

Furthermore, a (RKKK)₃ sequence, or other variations of this sequence,can be inserted into p193NS F5F2K. This sequence comprises, in part:

TCT AGA AAG AAG AAG CGC AAA AAA AAA [SEQ ID NO:77] Thr Arg Lys Lys LysArg Lys Lys Lys [SEQ ID NO:78] AGA AAG AAG AAG ACT AGT Arg Lys Lys LysThr Ser.

The sequence can be inserted with use of the 39-mer senseoligonucleotide (RKKK)₃(S) (i.e., comprising the sequence CT AGA AAG AAGAAG CGC AAA AAA AAAAGA AAG AAG AAG A [SEQ ID NO:79]), and the 39-merantisense oligonucleotide (RKKK)₃(a) (i.e., comprising the sequence CTAGT CTT CTT CTT TCT TTT TTT TTT GCG CTT CTT CTT T [SEQ ID NO:80]). Theresultant plasmid p193NS F5F2K(RKKK)₃ is depicted in FIG. 19. Thisplasmid also is known as p193NS F5F2K(RKKK3), p193 F5F2K(RKKK3), or p193F5FK(RK33). The relevant portion of the modified loop of the fiber knobpresent in p193 F5F2K(RKKK)₃ is:

CTT AAT GGC ACT AGA AAG AAG AAG CGC [SEQ ID NO:33] Leu Asn Gly Thr ArgLys Lys Lys Arg [SEQ ID NO:34] AAA AAA AAA AGA AAG AAG ACT AGT GAA LysLys Lys Arg Lys Lys Thr Ser Glu TCC ACA Ser Thr.

EXAMPLE 9

This example describes the construction of plasmids containing chimericpenton base proteins comprising UTV or UTV-like sequences.

The transfer plasmid pACT (ΔRGD) (also described as plasmid pAT in U.S.Pat. No. 5,559,099) was derived, in part, by manipulating a plasmidcontaining the unique BamHI/PmeI fragment (13259-21561) of the Ad5genome, and contains, among other things, a penton base proteincomprising a deletion of 8 amino acids constituting the α_(v) integrinbinding domain, and a substitution of the deleted region for amino acidsconstituting a unique SpeI site, for the convenient insertion ofexogenous sequences.

To construct plasmid pACT (RKKK)₃ (also known as pACT (RKKK3) or pACT(RK33)) depicted in FIG. 20, the complementary overlappingoligonucleotides RK33s and RK33a were directly ligated into aSpeI-digested pACT (ΔRGD) plasmid. Verification of thecorrectly-oriented clone was performed by PCR using the RK33a primer forthe plasmid and the upstream sense oligonucleotide primer A5s15002. Theplasmid also was verified to contain the correctly oriented insert bysequencing the DNA sequence in the region of the insert using theA5s15002 primer. The relevant portion of the UTV domain that will resultin the chimeric penton base protein in pACT (RKKK)₃ is:

AAC GAT ACT AGA AAG AAG AAG CGC AAA [SEQ ID NO:35] Asn Asp Thr Arg LysLys Lys Arg Lys [SEQ ID NO:36] AAA AAA AGA AAG AAG AAG ACT AGT GCC LysLys Arg Lys Lys Lys Thr Ser Ala ACA Thr.

The plasmid pACT (RK32) (which also can be called pACT (RKKK2) or pACT(RKKK)₂) depicted in FIG. 21 similarly can be constructed using theRK32s and RK32a overlapping primers. The relevant portion of the UTVdomain present in the chimeric penton base protein in pACT (RKKK)₂ is:

AAC GAT ACT AGA AAG AAG AAG AGA AAG [SEQ ID NO:37] Asn Asp Thr Arg LysLys Lys Arg Lys [SEQ ID NO:38] AAG AAG ACT AGT GCC ACA Lys Lys Thr SerAla Thr.

EXAMPLE 10

This example describes the construction of plasmids containing UTV orUTV-like sequences in the adenovirus hexon protein, and particularlywhich contain these sequences in an exposed loop of the adenovirus hexonprotein.

These plasmids can be constructed making use of another transferplasmid, plasmid pACT H11, depicted in FIG. 22. Plasmid pACT H11 itselfis derived from plasmid pACT (comprising from 13259-21561 of the Ad5genome), which contains the majority of the hexon protein codingsequence (corresponding to about 18842-21700). In particular, pACT H11can be constructed by incorporating an XbaI site into the loop 1 regionof the Ad5 hexon protein. Similar techniques can be used to incorporatean XbaI site, or any other convenient restriction site, into either theloop 1 or the loop 2 region, or into another exposed loop of the hexonprotein. Sense and antisense primers can be used to amplify the loop 1region from Ad5 DNA by PCR, and at the same time introduce a mutationwhich results in a unique mutated XbaI site in the loop 1 region.

In particular, the sense primer, GGACAGGGGCCCTACTTTTAAGCCCTACTCTGGCA[SEQ ID NO:81], containing the naturally occurring unique restrictionsite ApaI that occurs in pACT, and the antisense primer,ATCTTCACTGTACAATACCACTTTAGGAGTCAAGTTATCACCTCTAGATGCGGT CGCCT [SEQ IDNO:82], containing the unique restriction site, BsrGI, can be employed.The PCR product, containing the XbaI site can then be cut with BsrGI andApaI, and cloned back in to pACT to replace the ApaI to BsrGI fragment.The resultant plasmid, pACT H11, contains a unique XbaI site for theinsertion of UTV sequences into loop 1 of the hexon. The presence of theXbaI site in the pACT H11 clone can be verified by restriction digestionusing XbaI, which should linearize the plasmid.

Part of the unmutated hexon loop 1 amino acid sequence comprises thesequence TEATGNGDNL [SEQ ID NO:83]. In comparison, the mutated hexonloop 1 amino acid sequence following the wild-type TEA residues in pACTH11 (FIG. 22) comprises the sequence TASRGDNL [SEQ ID NO:40] (i.e.,encoded by the nucleic acid sequence ACCGCATCTAGAGGTGATAACTTG [SEQ IDNO:39]).

The XbaI site of pACT H11 then can be used as a unique site in which toclone universal targeting sequences such as RKKKRKKK [SEQ ID NO:74], forinstance, using the overlapping oligonucleotides, RK32s and RK32a. Theparticular plasmid that results from such manipulations, i.e. pACT H11(RKKK)₂ (or pACT H11 (RK32) or pACT H11 (RKKK2)) is depicted in FIG. 23.This plasmid comprises the sequence:

ACC GCA TCT AGA AAG AAG AAA CGC AAA AAG AAG ACT AGA [SEQ ID NO:41] ThrAla Ser Arg Lys Lys Lys Arg Lys Lys Lys Thr Arg [SEQ ID NO:42] GGT GATAAC TTG Gly Asp Asn Leu.

Other UTV or UTV-like sequences also can be cloned in the loop 1 regionof the hexon protein, and/or into the loop 2 region of the hexonprotein. For instance, a similar approach can be used to mutate thesequence encoding the hexon loop 2 to make plasmid pACT H12 (not shown)that contains a unique restriction site (such as a XbaI site) into whichfurther UTV sequences can be cloned.

Also, plasmid pACT H11 (RKKK)₃ (or pACT H11 (RKKK3) or pACT H11 (RK33))can be constructed making use of the complementary overlappingoligonucleotides RK33s and RK33a, and directly ligating the PCR productinto the XbaI-digested pACT H11 plasmid. Verification of thecorrectly-oriented clone can be performed by PCR using the RK32 senseprimer for the plasmid and an appropriate downstream antisenseoligonucleotide primer. The plasmid can be verified to contain thecorrectly oriented insert by sequencing the DNA sequence in the regionof the insert using the downstream antisense primer. Similar approachescan be employed for construction of analogous transfer vectors,particularly the analogous transfer vectors pACT H12 (RKKK)₂ and pACTH12 (RKKK)₃.

EXAMPLE 11

This example describes the construction of a plasmid having ashort-shafted fiber protein. In particular, this example describes theconstruction of the plasmid, p193 FSF9sK.

The plasmid p193 F5F9sK (also known as p193 F5F9K-Short) is depicted inFIG. 24. This vector encodes a chimeric fiber protein whereinapproximately two thirds of the Ad5 fiber shaft is deleted and the Ad5fiber knob is replaced with the Ad9 fiber knob.

The plasmid p193F5F9K-short was constructed from p193NS (F5*). Theoligonucleotide primers GGACTAGTAG CATTTAATAA AAAAGAAGAT AAGCGC [SEQ IDNO:84] and CCGGATCCTC ATTCTTGGGC GATATAGG [SEQ ID NO:85] were used toamplify the Ad9 sequence encoding the last shaft repeat and knob fromthe fiber gene. The PCR product was then purified, using standardtechniques, and digested with the restriction enzymes NheI and BamHI,which allowed cloning of the PCR product into the NheI/BamHI region ofthe p193NS (F5*) transfer plasmid. The resultant short-shafted fiberprotein can be employed in construction of adenoviral vectors, asdescribed below. Furthermore, any one or more of the aforementioned UTVor UTV-like sequences can be incorporated into the short-shafted fiber,and the resultant fiber can be employed for cell delivery.

EXAMPLE 12

This example describes the construction of plasmids containing UTV orUTV-like sequences in an extended structure, particularly in hexonand/or penton base protein, so as to result in lengthened hexon and/orpenton base proteins that accordingly are better able to contact cellsand participate in cell targeting. The resultant chimeric proteins are“spiked” in the sense that they comprise an insertion of a normativeamino acid sequence that will jut out from the virus surface.

The primers 1 alpha(s),GGGCTGCAGGCGGCCGCAGAAGCTGAAGAGGCAGCCACACGGGCTGAGGAGA A [SEQ ID NO:86],and 1 alpha(a), GGGGTGCACACAGCTTCGGCCTTAGCGTTAGCCTGTTTCTTCTGAGGCTTCTCGACCT [SEQ ID NO:87], can be used to amplify the region of the penton geneencoding the 32 amino acid α-helical domain that follows the RGDsequence. This 32 amino acid sequence comprises the sequenceATRAEEDRAEAEAAAEAAAPAAQPEVEKPQKK [SEQ ID NO:88]. The primers used alsocan encode an additional α-helical sequence on either end, such that,for instance, the final amplified DNA sequence encodes the sequence:

CTG CAG GCG GCC GCA GAA GCT GAA GAG GCA GCC ACA CGG GCT GAG [SEQ IDNO:89] Leu Gln Ala Ala Ala Glu Ala Glu Glu Ala Ala Thr Arg Ala Glu [SEQID NO:90] GAG AAG CGC GCT GAG GCC GAA GCA GCG GCC GAA GCT GCC GCC CCCGlu Lys Arg Ala Glu Ala Glu Ala Ala Ala Glu Ala Ala Ala Pro GCT GCG CAACCC GAC GTC GAG AAG CCT CAG AAG AAA CAG GCT AAC Ala Ala Gln Pro Glu ValGlu Lys Pro Gln Lys Lys Gln Ala Asn GCT AAG GCC GAA GCT GTG CAG GCG GCCGCA GAA GCT GAA GAG GCA Ala Lys Ala Glu Ala Val Gln Ala Ala Ala Glu AlaGlu Glu Ala GCC ACA CGG GCT GAG GAG AAG CGC GCT GAG GCC GAA GCA GCG GCCAla Thr Arg Ala Glu Glu Lys Arg Ala Glu Ala Glu Ala Ala Ala GAA GCT GCCGCC CCC GCT GCG CAA CCC GAG GTC GAG AAG CCT CAG Glu Ala Ala Ala Pro AlaAla Gln Pro Glu Val Glu Lys Pro Gln AAG AAA CAG GCT AAC GCT AAG GCC GAAGCT GTG CAC Lys Lys Gln Ala Asn Ala Lys Ala Glu Ala  Val Hiswhere the emboldened sequence corresponds to the nonpenton sequenceencoded by the primers, and the underlined sequence represents the aminoacids encoded by the two compatible restriction sites, SfcI and ApaLI.These amino acids also preserve the integrity of an alpha helixaccording to standard computer programs designed to predict α helixstructure.

The PCR product encoding these amino acids can be cut with both SfcI andApaLI, religated, and then cut again with both enzymes. Ligation oflike-site to like-site preserves the site for recutting; however,ligation of the compatible, but unlike sites, destroys the site.Therefore, upon recutting of the ligated product, multiple fragmentswill be produced which are multiples of the original size of the PCRproduct. There will be completely recut fragments (approximately 150bp), approximately 300 bp fragments (having one restriction sitedestroyed), and approximately 450 bp fragment (having 2 sitesdestroyed), and so on. The procedure accordingly allows the originalsequence encoding 50 amino acids (1 alpha) to be doubled (2alpha),tripled (3alpha), and so on, for cloning large, uninterrupted α-helicalregions into a protein to create a larger “spike”, or extension of theprotein.

For instance, the 2alpha double product (i.e., 2alpha2) can be clonedinto the first Ppu10I site of the plasmid, pSPdelta (depicted in FIG.25), to create the plasmid pSP2alpha (depicted in FIG. 26). The plasmidpSPdelta is constructed from the base plasmid pUC19, or any othersuitable cloning plasmid. The pSPdelta transfer plasmid can be employedto make further modifications of the penton or hexon protein that allowthe UTV sequence (or any other targeting sequence) to be elevated outfrom the virion surface. This elevation of the targeting sequence in aspiked structure (e.g., a type of “tower”) will minimize sterichindrance of the penton and hexon interaction with the cell surface bythe fiber protein, and will allow greater access of the chimeric pentonand hexon proteins for interacting with the cell surface.

The plasmid pSPdelta contains a unique Spel cloning site for theincorporation of UTV sequences. The SpeI cloning site is flanked oneither side by Ppu10I cloning sites which allow the incorporation of DNAsequences encoding amino acids that will elevate the UTV sequence awayfrom the virion surface. The pSPdelta plasmid was constructed by cloninginto the unique XbaI site of pUC19 overlapping oligonucleotides whichare designed to insert directly into the XbaI site. In particular, thesense oligonucleotide is:CTAGAGCAGCTATGCATGAAGGGACTAGTGGAGAGATGCATGCAGCCT [SEQ ID NO:91]. Theantisense, complementary oligonucleotide is:CTAGAGGCTGCATGCATCTCTCCACTAGTCCCTTCATGCATAGCTGCT [SEQ ID NO:92]. Theoligonucleotides are mixed in equimolar ratios and cloned into the XbaIsite of pUC19. The presence of the correct insert can be confirmed bysequencing across the region of the insert, and by cutting the plasmidwith Ppu10I. The XbaI sites on either side of the insert allow theconvenient removal of this section in later clones.

Because there are two Ppu10I sites in pSPdelta, the plasmid can bepartially restricted upon digestion with Ppu10I so that only a singlesite is cut. Ligation of the PCR product comprising the ApaLI and SfcIsites into the compatible Ppu10I site will destroy the first Ppu10I sitein the plasmid and also destroy the ApaLI and SfcI sites. Thesedestroyed sites are represented in FIG. 26 (showing the pSP2alpha2plasmid) by a “j”. A second doublet product then can be cloned into theremaining Ppu10I site of the pSP2alpha plasmid to produce the plasmidpSP2alpha2 depicted in FIG. 27, which contains an SpeI site forinsertion cloning of a UTV sequence, or other similar targetingsequence. Computer analysis of the secondary structure of theanticipated 2alpha2 protein confirms that it will be a complete α helixexcept for in the region of the SpeI cloning site.

Thus whereas pSPdelta comprises the sequence: TCT AGA GCA GCT ATG CATGAA GGG ACT AGT GGA GAC ATG CAT GCA [SEQ ID NO:43] Ser Arg Ala Ala MetHis Glu Gly Thr Ser Gly Glu Met His Ala [SEQ ID NO:44] GCC TCT AGA AlaSer Arg, pSP2alpha comprises the sequence: TCT AGA GCA GCT ATG CAG GCGGCC GCA GAA GCT GAA GAG GCA [SEQ ID NO:45] Ser Arg Ala Ala Met Gln AlaAla Ala Glu Ala Glu Glu Ala [SEQ ID NO:46] GCC ACA CGG GCT GAG GAG AAGCGC GCT GAG GCC GAA GCA GCG Ala Thr Arg Ala Glu Glu Lys Arg Ala Glu AlaGlu Ala Ala GCC GAA GCT GCC GCC CCC GCT GCG CAA CCC GAG GTC GAG AAG AlaGlu Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu Lys CCT CAG AAG AAA CAGGCT AAC GCT AAG GCC GAA GCT GTG CAG Pro Gln Lys Lys Gln Ala Asn Ala LysAla Glu Ala Val Gln GCG GCC GCA GAA GCT GAA GAG GCA GCC ACA CGG CCT GAGGAG Ala Ala Ala Glu Ala Glu Glu Ala Ala Thr Arg Ala Glu Glu AAG CGC GCTGAG GCC GAA GCA GCG GCC GAA GCT GCC GCC CCC Lys Arg Ala Glu Ala Glu AlaAla Ala Glu Ala Ala Ala Pro GCT GCG CAA CCC GAG GTC GAG AAG CCT GAG AAGAAA CAG GCT Ala Ala Gln Pro Glu Val Glu Lys Pro Gln Lys Lys GLn Ala AACGCT AAG GCC GAA GCT GTG CAT GAA GGG ACT AGT GGA GAG Asn Ala Lys Ala GluAla Val His Glu Gly Thr Ser Gly Glu ATG CAT GCA GCC TCT AGA Met His AlaAla Ser Arg, and pSP2alpha2 comprises the sequence: TCT AGA GCA GCT ATGCAG GCG GCC GCA GAA GCT GAA GAG GCA GCC [SEQ ID NO:47] Ser Arg Ala AlaMet Gln Ala Ala Ala Glu Ala Glu Glu Ala Ala [SEQ ID NO:48] ACA CGG GCTGAG GAG AAG CGC GCT GAG GCC GAA GCA GCG GCC GAA Thr Arg Ala Glu Glu LysArg Ala Glu Ala Glu Ala Ala Ala Glu GCT GCC GCC CCC GCT GCC CAA CCC GAGGTC GAG AAG CCT CAG AAG Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu LysPro Gln Lys AAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAG GCG GCC GCA GAALys Gln Ala Asn Ala Lys Ala Glu Ala Val Gln Ala Ala Ala Glu GCT GAA GAGGCA GCC ACA CGG GCT GAG GAG AAG CGC GCT GAG GCC Ala Glu Glu Ala Ala ThrArg Ala Glu Glu Lys Arg Ala Glu Ala GAA GCA GCG GCC GAA GCT GCC GCC CCCGCT GCG CAA CCC GAG GTC Glu Ala Ala Ala Glu Ala Ala Ala Pro Ala Ala GlnPro Glu Val GAG AAG CCT CAG AAG AAA CAG GCT AAC GCT AAG GCC GAA GCT GTGGlu Lys Pro Gln Lys Lys Gln Ala Asn Ala Lys Ala Glu Ala Val CAT GAA GGGACT AGT GGA GAG ATG CAG GCG GCC GCA GAA GCT GAA His Glu Gly Thr Ser GlyGlu Met Gln Ala Ala Ala Glu Ala Glu GAG GCA GCC ACA CGG GCT GAG GAG AAGCGC GCT GAG GCC GAA GCA Glu Ala Ala Thr Arg Ala Glu Glu Lys Arg Ala GluAla Glu Ala GCG GCC GAA GCT GCC GCC CCC GCT GCG CAA CCC GAG GTC GAG AAGAla Ala Glu Ala Ala Ala Pro Ala Ala Gln Pro Glu Val Glu Lys CCT CAG AAGAAA CAG GCT AAC GCT AAG GCC GAA GCT GTG CAG GCG Pro Gln Lys Lys Gln AlaAsn Ala Lys Ala Glu Ala Val Gln Ala GCC GCA GAA GCT GAA GAG GCA GCC ACACGG GCT GAC GAG AAG CGC Ala Ala Glu Ala Glu Glu Ala Ala Thr Arg Ala GluGlu Lys Arg GCT GAG GCC GAA GCA GCG GCC GAA GCT GCC GCC CCC GCT GCG CAAAla Clu Ala Glu Ala Ala Ala Glu Ala Ala Ala Pro Ala Ala Gln CCC GAG GTCGAG AAG CCT CAG AAG AAA CAC GCT AAC GCT AAG GCC Pro Glu Val Clu Lys ProGln Lys Lys Gln Ala Asn Ala Lys Ala GAA GCT GTG CAT GCA GCC TCT AGA GluAla Val His Ala Ala Ser Arg.

Targeting sequences such as UTV or UTV-like sequences can be cloned intothe SpeI site of the plasmid pSP2alpha2. In particular, the RK32s andRK32a overlapping oligonucleotides can be cloned into the SpeI site tocreate pSP2alpha2 (RKKK)₂ (or, pSP2alpha2 (RK32) or pSPSalpha2 (RKKK2)).The plasmid pSP2alpha2 (RKKK)₃ (or, pSP2alpha2 (RK33) or pSPSalpha2(RKKK3)) can be similarly constructed. Alternately, the entire 2alpha2α-helical domain can be removed from the plasmid by restriction withXbaI and cloned into the compatible SpeI site of pACT (ΔRGD) to createpACT 2alpha2 (RKKK)₂ (which also can be called pACT 2alpha2 (RKKK2) orpACT 2alpha2 (RK32)). Similar techniques can be employed to produce pACT2alpha2 (RKKK)₃ (which also can be called pACT 2alpha2 (RKKK3) or pACT2alpha2 (RK33)).

Similarly, chimeric hexon proteins that are spiked (i.e., comprisesequences resulting in their extension) can be constructed by cloningthe 2alpha2 α-helical domain into the XbaI site of pACT H11 to createpACT H11 2alpha2 (RKKK)₂ (which also can be called pACT H11 2alpha2(RKKK2) or pACT H11 2alpha2 (RK32)). Similar techniques can be employedto produce pACT H12 2alpha2 (RKKK)₂ (which also can be called pACT H122alpha2 (RKKK2) or pACT H12 2alpha2 (RK32)).

EXAMPLE 13

This example describes the construction of further adenoviral vectors,in addition to those previously described, which contain UTV or UTV-likesequences in the adenoviral fiber protein.

Construction of adenovirus vectors containing UTV modifications in thefiber can be accomplished in multiple ways by those skilled in the art.One method to create the UTV fiber vectors from plasmids described aboveis to first linearize the plasmid DNA with SalI and then transfect thisDNA into a 293 packaging cell line that was infected just prior totransfection with an E4-deleted adenovirus. E4-deleted adenovirusvectors are incapable of replicating in cell lines such as the 293 cellline, which only provide the adenovirus E1 regions in trans.Recombination of the plasmids which contain the modified fiber gene andthe E4 regions with the E4-deleted DNA results in areplication-competent, E4-containing vector which carries the modifiedfiber gene.

Accordingly, the plasmids p193NS (F5*) pGS(K7), pBSS 75-100 pGS(null),pBSS 75-100 pGS(RKKK)₂, pBSS 75-100 pGS(RKKK)₃, pBSS 75-100 pGS(tat),p193NS F5F2K(RK32), and p193 F5F9sK were each linearized with SalI andtransfected into 293 cells infected 1 hour prior with either theE4-deleted adenovirus vector, GV11 A.Z (which carries the LacZ geneunder the control of a cytomegalovirus (CMV) promoter), or GV11A.S(which carries a secretory alkaline phosphatase gene under the controlof a CMV promoter). The resultant adenovirus vectors, AdZ.F(pK7),AdZ.F(pGS), AdZ.F(RKKK)₂ (also known as AdZ.F(RKKK2) or AdZ.F(RK32)),AdZ.F(RKKK)₃ (also known as AdZ.F(RKKK3) or AdZ.F(RK33)), AdZ.F(tat),AdZ.F5F2K(RKKK)₂ (also known as AdZ.F5F2K(RKKK2) or AdZ.F5F2K(RK32)),and AdZ.F5F9sK (also known as AdZ.F5F9K-Short) were obtained and werepurified through two successive rounds of plaquing on 293 cells.

All the vectors were verified to contain the correct sequence throughPCR across the region of the insert, and by restriction analysis ofviral DNA obtained from vector-infected 293 cells by Hirt extraction.Western analysis (as previously described) also can be employed toexamine protein size, if so desired. Western analysis of fiber proteinfrom vector particles and/or vector-infected cell lysateselectrophoresed on a polyacryamide gel should show a corresponding shiftin the mobility of the fiber protein compared to unmodified fiberprotein that is consistent with the presence of additional amino acidsequences. For instance, Western analysis of AdZ.F(pK7) particlesverified that its fiber protein is shifted up compared to that of theAdZ vector comprising unmodified fiber, consistent with the presence ofadditional amino acids in the AdZ.F(pK7) fiber protein.

Other plasmid maps depicted herein similarly can be made into adenoviralvectors by utilizing the same procedure outlined above (or minorvariations thereof).

EXAMPLE 14

This example describes the construction of adenoviral vectors whichcontain UTV or UTV-like sequences in the adenoviral penton base protein.

The method of making an adenoviral vector comprising a chimeric pentonbase protein is described, for instance, in Wickham et al., J. Virol.,70, 6831-6838 (1996). A pACT vector described above containing thechimeric penton base protein (e.g., transfer plasmids pACT 2alpha2(RKKK)₂, pACT H12 2alpha2 (RKKK)₂, and pACT (ΔRGD)) can be digested, forinstance, with BamHI, to linearize the plasmid. Ad5 DNA can be digestedwith the restriction endonuclease XmnI, which cuts wild-type Ad5 atpositions 14561 and 15710 within the Ad5 genome. The two largerfragments are purified away from the smaller 1 kb piece then transfectedalong with the linearized plasmid into the appropriate cell line (e.g.,a 293 cell line) to produce recombinant virus.

Adenoviral vectors produced in this fashion are purified frompotentially-contaminating unmodified vectors through two successiverounds of plaque purification on 293 cells. The resultant vectors arethen verified to contain the correct sequence in the penton base regionthrough restriction analysis of viral DNA obtained following Hirtextraction of vector-infected 293 cells. Sequencing of PCR productsgenerated by amplifying the region of the insert from the viral DNA canalso be used to verify the presence of the insert. Western analysis ofthe chimeric penton base electrophoresed on a polyacrylamide gel shouldshow a corresponding shift in the mobility of the penton base comparedto unmodified penton base that is consistent with the presence ofadditional amino acid sequences in the chimeric protein.

EXAMPLE 15

This example describes the construction of adenoviral vectors containingUTV or UTV-like sequences in the hexon protein.

To construct the virus AdZ.H(RKKK)₂, left and right vector arms areprepared that contain DNA sequences which overlap and will recombine oneither side of the PmeI to BamHI pACT H11 (RK32) sequence to create anintact AdZ.H (RK32) genome. To construct the left arm, purified Ad5 DNAis restriction digested with AgeI, which cuts Ad5 at positions 14499,15283, 19017, 23063, 23656, 23411, and 31102. The 0-14499 fragment thencan be employed as the left arm and purified from the other fragments bygel electrophoresis. The right arm can be prepared by digesting Ad5 DNAwith DrdI. DrdI cuts Ad5 at positions 5458, 7039, 14004, 15593, 17257,and 21023. The 21023-35938 bp fragment then can be used as the right armand purified from the other fragments by gel electrophoresis. These twofragments are then transfected with the PmeI/BamHI fragment from pACTH11(RK32) into 293 cells. PmeI cuts at position 13258 in Ad5, and BamHIcuts at position 21562 in Ad5. Similar techniques can be employed toproduce AdZ.H(RKKK)₃.

EXAMPLE 16

This example describes the construction of vectors containing ashort-shafted fiber protein.

The method described herein for construction of an adenovirus from thetransfer plasmid p 193 FSF9Kshort can similarly be employed for theconstruction of other adenoviral vectors from other short-shaftedfibers. Namely, the transfer plasmid p 193 F5F9Kshort, which containsthe essential E4 region of adenovirus, was cut with SalI and transfectedinto 293 cells, which had been infected one hour earlier with theadenoviral vector AdSE.E4Gus. AdSE.E4Gus lacks the E4 region of theadenoviral genome, and cannot replicate in 293 cells in the absence ofcomplementation for the E4 genes. Thus, only when the AdSE.E4Gus DNArecombines with the p193 F5F9K short plasmid DNA to obtain the E4 genesis the vector able to replicate in 293 cells. During this recombinationevent, the newly formed vector also acquires the mutated fiber proteincoding sequences encoded by the plasmids. Viable recombinant E4⁺adenovirus containing the F5F9Kshort fiber chimera were then isolated byplaquing the transfected cell lysates 5 days after transfection. Theresultant vector AdSE.F5F9Kshort was isolated and purified by standardvirological techniques involving two successive rounds of plaquing on293 cells. The vector was verified to contain the correct insert by PCRand restriction analysis of viral DNA. Oligonucleotide primers, whichprime on either side of the fiber gene, confirmed that the PCR productwas of the correct size for that encoded by a shortened chimeric fibergene. Restriction analysis of the vector DNA showed that the new vectorcontained the correct restriction sites that are unique to the Ad9 fiberknob.

EXAMPLE 17

This example describes the construction of adenoviral vectors thatcontain short-shafted fiber proteins and chimeric penton base proteinsincorporating UTV or UTV-like sequences.

For this construction, AdS.F9sK viral DNA can be digested with XmnI, asdescribed above. The plasmid pACTH11 (RK32)is then cut with therestriction enzymes PmeI and BamHI. The restriction digested viral andplasmid DNAs are purified and transfected into 293 cells. The resultantvectors are isolated by two successive rounds of plaque purification on293 cells, and are verified to contain the correct sequence in thepenton base region by restriction analysis of viral DNA obtained fromvector-infected 293 cells by Hirt extraction.

Sequencing of PCR products generated by amplifying the region of theinsert from the viral DNA also can be used to verify the presence of theinsert. Western analysis of the chimeric penton base protein on apolyacrylamide gel should show a corresponding shift in the mobility ofthe chimeric penton base compared to unmodified penton base that isconsistent with the presence of additional nonnative amino acidsequences (and absence of native amino acid sequences).

Other plasmids for which maps are presented herein and which were notmade into an adenoviral vector can be made by utilizing the same or aslightly modified version of the procedure outlined above. Inparticular, the short-shafted fiber protein can be incorporated into anadenovirus having a “spiked” chimeric penton base protein thatfurthermore optionally can incorporate a UTV or UTV-like sequence.

EXAMPLE 18

This example describes the construction of adenoviral vectors thatcontain short-shafted fiber proteins and chimeric hexon proteinsincorporating UTV or UTV-like sequences.

Viral DNA can be isolated from a short-shafted vector such as AdZ.F9sKand cut with the restriction enzymes described above for making vectorscomprising UTV-containing chimeric hexon proteins. All other steps arethe same as described, for instance, in Example 17. The resultant vectorshould contain the short-shafted fiber protein and the chimeric hexonprotein incorporating UTV or UTV-like sequences. Moreover, this approachcan be employed with a variety of transfer plasmids comprising differentchimeric hexon proteins. In particular, the short-shafted fiber proteincan be incorporated into an adenovirus having a “spiked” chimeric hexonprotein that furthermore optionally can incorporate a UTV or UTV-likesequence.

EXAMPLE 19

This example describes an evaluation of vectors, particularly adenoviralvectors, according to the invention, which comprise UTV or UTV-likesequences.

For instance, to confirm that the addition of UTV or UTV-like sequenceshas no effect on virus assembly, the virus growth kinetics of thevectors can be assessed. As representative of a UTV-sequence containingplasmid, the growth behavior of pAd.F(pK7) was monitored and compared tothat of wild-type adenovirus (Ad5), as well as the adenoviral vectorAdZ.F(RGD), which contains an insertion of a RGD peptide motif presentin the sequence SACDCRGDCFCGTS [SEQ ID NO:93]. For these studies, 293cells were infected at a multiplicity of infection of 5 active virusparticles/cell with either Ad5, AdZ.F(RGD) or AdZ.F(pK7), and the numberof infectious particles (fluorescent focus units (FFU)) produced percell was determined following the harvesting of the cells at 1, 2, and 3days post infection. The titers of AdZ.F(RGD) or AdZ.F(pK7) weresomewhat lower, but not dramatically different than the titer of Ad5. Ascan be seen in FIG. 28, the peak titers of AdZ.F(RGD) and AdZ.F(pK7)were 80% and 56%, respectively, that of Ad5. These results confirm thatthe growth kinetics of the two vectors are not substantially affected bythe addition of sequences, particularly a UTV or UTV-like sequence, ontothe end of the fiber protein. The results also suggest that furthervectors comprising UTV or UTV-like sequences will not exhibit aberrantgrowth behaviour.

Furthermore, vectors containing UTV or UTV-like sequences can beevaluated for their ability to bind to cells or deliver genes to beinhibited by negatively-charged molecules (e.g., heparin, heparansulfate, chondroitan sulfate, etc.), soluble adenoviral coat proteins,or by pretreatment of cells with agents (e.g., chondroitinase,heparinase, sialidase, etc.) that cleave such negatively-chargedmoieties (see, e.g., Wickham et al., Nature Biotechnology, 14, 1570-1573(1996), as well as the preceding Examples). Soluble fiber protein willnot inhibit the majority of the binding of a UTV vector to a cell(Wickham et al. (1996), supra). These results suggest that theincorporation of UTV or UTV-like sequences into penton, hexon or fiberwill not impair the ability of a recombinant adenovirus containing thechimeric coat protein to effect gene delivery.

Also, studies of infection in vivo, or in vitro transfections done inthe presence of whole blood, can be employed to confirm that the UTVvectors of the present invention are not limited for systemic deliverydue to saturation of the polycations on the recombinant adenoviruseswith polyanions in the blood. In the event that such binding impedes thecapability of a particular virus for target cell transduction, the viruscan be administered in a higher dose, preferably with provision beingmade to reduce any immune response associated with such a higher dose(e.g., administration of another serotype of adenoviral vector, ortechniques described in PCT International Application WO 96/12406;Mastrangeli et al., Human Gene Therapy, 7, 79-87 (1996)).

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference to the same extent as if each reference were setforth in its entirety herein.

While this invention has been described with an emphasis upon preferredembodiments, it will be apparent to those of ordinary skill in the artthat variations in the preferred embodiments can be prepared and usedand that the invention can be practiced otherwise than as specificallydescribed herein. The present invention is intended to include suchvariations and alternative practices. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

1. A recombinant adenovirus comprising a penton base protein and a fiberprotein, wherein the penton base protein and fiber protein lack nativebinding to a cell surface binding site.
 2. The recombinant adenovirus ofclaim 1, wherein the penton base protein is unable to bind an α_(v)integrin receptor.
 3. The recombinant adenovirus of claim 2, wherein thepenton base protein lacks a native RGD sequence.
 4. The recombinantadenovirus of claim 1, wherein the fiber protein trimerizes.
 5. Therecombinant adenovirus of claim 1, wherein the fiber protein lacks thefiber knob.
 6. The recombinant adenovirus of claim 5, wherein the fiberprotein trimerizes.
 7. A recombinant adenovirus comprising (a) a pentonbase protein that lacks native binding to a cell surface binding siteand (b) a nonnative amino acid sequence that binds a cell surfacebinding site.
 8. The recombinant adenovirus of claim 7, wherein thepenton base protein is unable to bind an α_(v) integrin receptor.
 9. Therecombinant adenovirus of claim 7, wherein the penton base protein lacksa native RGD sequence.
 10. The recombinant adenovirus of claim 7,wherein the adenovirus further comprises a fiber protein lacking nativebinding.
 11. The recombinant adenovirus of claim 10, wherein the fiberprotein trimerizes.
 12. The recombinant adenovirus of claim 10, whereinthe nonnative amino acid sequence is inserted into the fiber protein orin place of at least a portion of the fiber protein.
 13. The recombinantadenovirus of claim 12, wherein the nonnative amino acid sequence isinserted into a knob region of the fiber protein or in place of at leasta portion of a knob region of the fiber protein.
 14. The recombinantadenovirus of claim 13, wherein the fiber protein trimerizes.
 15. Therecombinant adenovirus of claim 13, wherein the nonnative amino acidsequence is inserted into an exposed loop of the adenovirus fiber knobor in place of at least a portion of an exposed loop of the adenovirusfiber knob.